Inflammation is triggered immediately after cardiac injury, and it has been hypothesized to promote regeneration in regenerative models such as zebrafish.  Myeloid differentiation factor 88 (MyD88) is a central adaptor molecule for the Toll-like receptor and interleukin-1 receptor signaling pathways, both of which are essential regulators of inflammation.  However, how specific molecules, such as MyD88, regulate inflammation after cardiac injury remains unclear.  In this project, we are testing the hypothesis that the MyD88 signaling axis plays a central role in the initiation of the inflammatory response after injury and that this early response is required for cardiac regeneration in zebrafish.  Our data indicate that zebrafish lacking functional MyD88 display features linked to impaired cardiac regeneration potential, including reduced immune cell recruitment to the injured tissue and reduced coronary endothelial cell proliferation at the border zone.  Additionally, transcriptomic analysis reveals that myd88 mutants exhibit a downregulation of immune-related processes after cardiac cryoinjury.  To understand the mechanisms activated downstream of MyD88, we identified effectors of the MyD88 signaling pathway and are studying their role with gain- and loss-of-function tools.  To determine the cell types in which MyD88 signaling plays a role during cardiac regeneration, we are using a transgenic approach to block MyD88 function in a cell type-specific manner.  This project will provide insights into the cellular and molecular mechanisms involved in the inflammatory response during zebrafish cardiac regeneration.  By modulating specific immune components and their response to injury, we could potentially improve the regeneration outcome in mammalian models including in humans.

The embryonic epicardium undergoes epithelial-to-mesenchymal transformation, contributing cells for formation of coronary vasculature. It is also a source of paracrine cues that are essential for fetal cardiac growth, coronary vessel patterning, and regenerative heart repair.

We have modified an established model of early embryonic transmural myocardial cryoinjury (resulting in incomplete repair by smooth muscle) by performing it at the stage, where the heart is already covered by the epicardium. We hypothesized that in the presence of the epicardium, the mechanisms of reparation will be different, and a complete regeneration will occur similar to adult fish or mammalian neonatal models.

Myocardial injury was induced by cryoprobe on ventricle of avian embryos (chicken and quail) at Hamburger-Hamilton (HH) stage 21, which were then re-incubated until HH31. Myocardial regeneration was analyzed electrophysiologically by epicardial optical mapping, and morphologically by immunohistochemical markers for endothelium (QH1), myocytes (MF20), and smooth muscle actin (SMA).

Functional analysis showed changes in epicardial signal spreading in the cryoinjured area. There were profound changes in the epicardium, which was thickened, detached and wrinkled in the affected, but also in the remote area. Numerous newly formed microvessels (QH1, SMA) and myocytes (MF20-positive) were present in the subepicardial region, confirming the regenerative capability.

In conclusion, embryonic myocardial cryoinjury at trabeculated stage induces epicardial activation with enhanced vascularization and myocardial regeneration. This could be potentially used for devising novel regenerative strategies in humans that could use epicardial activation to repair myocardial infraction.

Shroom3 is a member of a small family of proteins that regulate cell shape by coordinating cell signaling and cytoskeletal architecture. Shroom3 is comprised of a PDZ domain that facilitates localization, and two ASD domains that mediate actin and myosin dynamics. Through mouse and human studies, Shroom3 mutations have been implicated in congenital heart defects, suggesting that Shroom3 may be important in cardiac development. In the heart, shroom3 is primarily expressed in cardiomyocytes (CMs). Using CRISPR/Cas9 technology, we generated a global homozygous null mutation (shroom3^-/-) in zebrafish that survive to adulthood in contrast to the embryonic lethal gene-trap mutagenesis observed in mice. Differential gene expression analysis, ascertained by from wildtype versus shroom3^-/- zebrafish whole heart RNAseq, revealed significant changes in several signaling pathways known to regulate cardiac regenerative capacity including Hippo, PI3K-Akt, HIF-1, and ErbB in addition to metabolic alterations indicative of heart failure development. Although external heart morphometric analysis revealed no obvious differences, shroom3^-/- maturation is delayed overall compared to wildtypes. Functional cardiovascular assessment through an intermittent-flow variable swim tube fitness test illustrated that mutants are unable to metabolize oxygen efficiently and fatigue faster than wildtype counterparts. These findings were further supported via high frequency echocardiography which showed that shroom3^-/- had a statistically lower stroke volume, ejection fraction, and fractional shortening. Finally, an impaired regenerative response was observed in mutants at both 7 and 30 days post ventricular cryoinjury. These preliminary findings suggest that Shroom3 likely contributes to cardiac function and response to injury in zebrafish.

The characterization of novel mechanisms safeguarding cell fate identity in differentiated cells is essential for 1) our understanding of how differentiation is maintained in healthy tissues, and 2) the development of new strategies to improve therapeutic reprogramming. In this context, we recently identified a set of four transcription factors (TFs) that collectively promote cell fate stability and restrict direct lineage reprogramming in differentiated cells. Among those factors, JUNB was found to oppose reprogramming by limiting reprogramming TF (i.e MEF2C) ability to access its target DNA and remodel chromatin. AP-1 TFs, such as JUNB, are known to mediate their function via homo and hetero-dimerization with other proteins, thus we hypothesized that yet to be identified JUNB-interacting proteins might cooperate with JUNB to regulate chromatin accessibility and promote cell fate stability. Here we describe the identification and characterization of H1F0 as a novel JUNB interactant mediating cell fate stability and opposing cardiac reprogramming.

Arrhythmias are a hallmark of myocardial infarction (MI) and contribute to poor prognosis. Increasing evidence implicates the ventricular conduction system (VCS) in generating arrhythmias. How cardiac injury affects the global architecture and cellular composition of the VCS, and whether the VCS can endogenously regenerate or repair itself, have not been systematically described. The neonatal mouse heart can regenerate following left anterior descending coronary artery ligation at postnatal day 1 (P1). The P1 heart replaces lost cardiomyocytes, removes temporary scarring, and recovers full function. This transient regeneration capacity is lost during the first week, such that P7 hearts fail to regenerate myocardium and instead maintain a permanent fibrotic scar.

We used the knock-in reporter line Cx40-eGFP to investigate VCS changes after injury in P1 versus P7 mice. By developing advanced tissue-clearing and wholemount imaging, we determined the 3D anatomy of the postnatal VCS. We observed loss of VCS cells near the injury site in infarcted P1 and P7 hearts, resulting in discontinuity within the Purkinje network. Longitudinal analysis showed an increased density of connexin-40-positive cells surrounding the injury zone at P1. We are currently using single-cell transcriptomic analysis to identify molecular pathways associated with VCS response to injury, (re-) growth and function. Collectively, our work identifies specific morphological changes in the Purkinje network following MI across the regenerative window, with evidence of recovery at early neonatal stages. These findings provide significant insights into VCS pathophysiology during heart injury and may identify molecular targets to maintain synchronous contraction in infarcted adult hearts.

Bmp and Fgf signaling are widely involved in multiple aspects of embryonic development. Non-coding RNAs, such as microRNAs, have also been recently reported to play essential roles during embryonic development. We previously demonstrated that microRNAs are capable of modulating cell fate decision during proepicardial/ septum transversum (PE/ST) development in chicken, since over-expression of miR-23 blocked while miR-125, miR-146, miR-223 and miR-195 enhanced PE/ST-derived cardiomyogenesis, respectively. Importantly, regulation of these microRNAs is distinct modulated by Bmp and Fgf administration. In this study, we aim to dissect the functional role of Bmp and Fgf signaling during mouse ST/PE development, their implication regulating these microRNAs and their impact on lineage determination. Mouse PE/ST explants and epicardial/endocardial cell cultures were distinctly administrated Bmp and Fgf family members. qPCR analyses of microRNAs, cardiomyogenic, fibrogenic differentiation markers as well as key elements directly epithelial to mesenchymal transition were evaluated. Our data demonstrate that neither Bmp2/Bmp4 nor Fgf2/Fgf8 signaling is capable of inducing cardiomyogenesis, fibrogenesis or inducing EMT in mouse ST/PE explants, yet upregulation of miR-146 and miR-195 is observed. RNAseq analyses in mouse PE/ST and embryonic epicardium identified novel Bmp and Fgf family members that might be involved in such cell fate differences, such as Bmp6, Bmp7, Bmp19, Fgf5, Fgf7 and Fgf10. Administration of these Bmp and Fgf family members in mouse PE/ST lead to limited modulation of EMT induction and cardiomyogenic and/or fibrogenic differentiation. Thus, our data support the notion of species-specific differences regulating PE/ST cardiomyogenic lineage commitment.

The Wilms tumor suppressor gene (Wt1) encodes a C2H2-type zinc-finger transcription factor that participates in transcriptional regulation, RNA metabolism and protein-protein interactions. WT1 is critically involved in the development of several organs, including kidneys and gonads, spleen, adrenals, liver, and diaphragm (Hastie, 2017). WT1 is highly expressed in the embryonic epicardium where it regulates a process of epicardial-mesenchymal transformation and the development of the epicardial-derived cells.

We have recently shown evidence of a transient Wt1 expression in about 25% of cardiomyocytes of mouse embryos. Conditional deletion of this expression in the cardiac troponin T lineage caused abnormal sinus venosus and atrium development, thin ventricular myocardium and, in some cases, interventricular septum and cardiac wall defects (Díaz del Moral et al., Front Cell Dev Biol. 2021;9:683861).

We aimed to know if Wt1 is also expressed in adult cardiomyocytes and what could be the consequences of its conditional deletion for cardiac homeostasis and/or in the response to damage induced by isoproterenol and doxorubicin treatments.  For conditional deletion of Wt1 in cardiomyocytes, we generated tamoxifen inducible Wt1 mutants by crossing aMHC^MerCreMer mice with homozygous Wt1 conditional mice, where the first exon of Wt1 is flanked by loxP sites.

We have found experimental evidence of a low expression of Wt1 in postnatal murine cardiomyocytes, using reporter and lineage tracing models as well as qPCR. Our preliminary data suggest that conditional deletion of Wt1 in cardiomyocytes induces interstitial fibrosis, increased oxidative stress markers, altered metabolism and mitochondrial dysfunction in Wt1-deficient cardiomyocytes. In addition, conditional deletion of Wt1 in adult cardiomyocytes increases the damage induced by doxorubicin and isoproterenol treatments. These findings suggest a novel role of Wt1 in myocardial physiology and protection against damage.

Cardiomyocyte renewal by dedifferentiation and proliferation has fuelled the field of regenerative cardiology in recent years, while the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterised by the restoration of function that is lost during dedifferentiation and is key to the healing process following injury. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: dedifferentiation, followed by redifferentiation. Here, using temporal RNAseq and proteomics, we survey the landscape of the dedifferentiation-redifferentiation process in the adult mouse heart. We find well characterised dedifferentiation pathways, such as reduced oxphos, increased proliferation and increased EMT-like features, largely return to normal, though surprisignly, elements of residual dedifferentiation remain, even after contractile function is restored. These hearts appeared rejuvenated and showed robust resistance to ischaemic injury. We find that redifferentiation is driven by negative feedback signalling, notably through LATS1/2 Hippo pathway activity. Disabling LATS1/2 in dedifferentiated cardiomyocytes augments dedifferentiation in vitro and prevents redifferentiation in vivo. Taken together, our data reveal the non-trivial nature of redifferentiation, whereby elements of dedifferentiation linger in a surprisingly beneficial manner. This cycle of dedifferentiation-redifferentiation protects against future insult, in what could become a novel prophylactic treatment against ischemic heart disease for at-risk patients.

Objectives

There is increasing evidence of the importance of interstitial cells in cardiovascular development and disease. In contrast to the adult mammalian heart, the adult zebrafish heart has the ability to regenerate. However, little is known about how interstitial cell populations contributing to this. We aim to better characterise interstitial cells with an emphasis on cardiac fibroblasts, during the regeneration process.

Methods

Using immunohistological analysis of the zebrafish heart from multiple transgenic reporter lines, we were able to quantify the various cell types that comprise the adult zebrafish heart. In addition, we performed single-cell RNA sequencing (scRNA-seq) on uninjured and amputated hearts at various time points in order to uncover the transcriptional properties of cardiac fibroblasts, as well as other interstitial cell populations that support adult zebrafish heart regeneration. The data was cross-referenced to published scRNA-seq data from murine studies to identify features of zebrafish cardiac fibroblasts.

Results

We were able to establish that, as in the adult mouse heart, endothelial cells and cardiomyocytes where the main cell types. However, fibroblasts where far less abundant in the adult zebrafish heart. Indeed, they represented 1-2% versus 11% of total cells in adult zebrafish versus mouse heart, respectively. ScRNA-seq analysis revealed features of interstitial cells that may support regeneration. Notably, in zebrafish, we found very limited fibroblast to αSMA⁺-myofibroblast transition as compared with injured mouse heart. By pharmacologically blocking regeneration, we found that αSMA⁺ expression was upregulated, but not in fibroblasts expressing high levels of collagen.

Conclusion

Our study suggests that features of myofibroblast transition, such as increased aSMA expression, are less pronounced in zebrafish cardiac fibroblasts as compared to fibroblasts in regenerating neonatal or infarcted adult mouse heart.

Myocardial infarction (MI) induces death of cardiac muscle and replacement by a non-contractile fibrotic scar, which ultimately leads to heart failure. Current treatments restore blood flow and assist with cardiac workload but none are regenerative, and clinical trials using stem-cell- based approaches have been disappointing. We have previously shown that the cardiac lymphatic system is activated following MI and that further stimulation with the lymphatic endothelial specific isoform of VEGF-C, VEGF-CC156S, resolves the immune response and improves cardiac function. Whilst this study demonstrates the potential of this approach, VEGF-CC156S is sub-optimal for clinical use. 

Our aim is to target the lymphatic vasculature to enhance the intrinsic regenerative potential of the infarcted adult heart using small molecules. To achieve this, we have established a sprouting assay using human lymphatic endothelial cells (hLEC), to mimic lymphangiogenesis in vitro and to provide the basis of a phenotypic screen. We have miniaturized this cell-based assay into a 384 well-plate format and have begun to screen focused libraries of compounds (epigenetic regulators, kinase inhibitors and stem cell modulators). Using automated imaging and post-hoc analyses we have selected, clustered and quantified hit molecules, which are currently being validated and further characterised through combined medicinal chemistry and preclinical models of adult cardiac injury.

Cardiac valve disease is a prevalent cause of morbidity and mortality worldwide, in which the cardiac valves that are normally ensuring unidirectional blood flow during the cardiac cycle, become defective. In severe cases, the diseased valve is surgically removed and replaced by decellularized implants, which generally rely on the repopulation by endogenous cells from the host. However, this recellularization process which ensures the stability and growth of the implant is greatly challenged in mammals due to the limited regenerative potential of their cardiac tissues.

We recently showed that adult zebrafish are capable of regenerating their cardiac valves. With the use of the nitroreductase-metronidazole genetic ablation system, we showed that valve-cell ablation in the adult zebrafish triggers the recruitment of endothelial cells and kidney marrow-derived cells, leading to the formation of a new functional valve. However, studying this process in histological sections of fixed tissues limits the assessment of the cellular contributions and rearrangements that take place during cardiac valve regeneration in vivo.

To overcome the limitations associated with adult specimens, we are now using zebrafish larvae to study the cellular dynamics and rearrangements involved in valve cell recruitment through live imaging. We have observed that also in larval stages, genetic ablation triggers a regenerative program leading to the recruitment of new valve cells, clearly visible at five days post-ablation. We also observed that this program is accompanied by an increase in the number of immune cells in the vicinity of the valve. We are now characterizing the different cellular sources and molecular pathways contributing to the formation of the new valve through live-imaging microscopy.

Overall, with this approach we expect to clarify the in vivo mechanisms of new valve cell recruitment after valve-cell ablation and determine the key factors promoting cardiac valve regeneration.

The mouse heart can regenerate during a short time window after birth, which coincides with maturation of the ventricular myocardium and compaction of trabeculae. These transient invaginations at the inner surface of the developing heart give rise to both contractile cardiomyocytes (CM) and Purkinje fibers of the ventricular conductive system (VCS). Purkinje fibers are recruited throughout embryonic and fetal development in successive waves, including a major wave of around the time of birth. This depends on maximal Nkx2-5 levels and Nkx2-5 heterozygous mice present a hypoplastic VCS. Here we investigate the contribution of trabecular myocardium to the regeneration of contractile and conductive cardiac tissues after neonatal myocardial infarction (MI). 

Neonatal MI was performed by permanent ligation of the left coronary artery on one-day-old mice. Trabecular derivatives were genetically traced using Connexin40 (Gja5) driven inducible Cre recombinase, revealing an active participation and increased proliferation of trabecular-derived cells during cardiac repair. Moreover, subendocardial myocardium re-expresses Cx40 under ischemia, consistent with a switch towards an immature phenotype. Surprisingly, we observed that regenerated hearts possess a global and permanent hyperplasia of the distal VCS despite spatially restricted damage following MI. This MI induced hyperplasia is not found in Nkx2-5+/- mice, suggesting that the underlying mechanism is Nkx2.5 dependent. We hypothesize that VCS hyperplasia under conditions of regeneration is an extension of the normal perinatal recruitment wave.

Together our data uncover cellular mechanisms involved in postnatal regeneration that resemble normal maturation of the ventricular myocardium. Trabecular-derived CM dedifferentiate toward an immature bipotent phenotype. The proliferation of these cells helps in repopulating the myocardial ischemic zone but produces an excess of PF which leads to VCS hyperplasia.

Myocardial infarction causes irreversible loss of cardiomyocytes and formation of a fibrotic scar, impairing cardiac function in patients. While the mammalian heart is incapable of regenerating the lost cardiomyocytes following injury, zebrafish can completely regenerate their heart through cell cycle re-entry of pre-existing cardiomyocytes. The molecular mechanisms underlying differences in regenerative capacity between species have yet to be unraveled. We employed spatial transcriptomics on mouse and zebrafish hearts post injury and compared the transcriptomes of their respective border zones to identify overlapping and diverging expression patterns. This dataset will be a helpful tool to identify drivers of cardiac regeneration. Here we will present the dataset and its validation. Furthermore, the dataset was used to select candidate genes for functional analysis in zebrafish using a knock-out approach of which the results will be presented here.

Dysregulated sympathetic innervation pattern and activity are observed in a number of cardiovascular diseases including post myocardial infarction.  Upon cardiac injury, sympathetic nerves (SNs) in the neonatal mouse heart migrate into the injury site and ablation of SNs post-injury impairs neonatal mouse heart regeneration.  In contrast, SNs fail to migrate into the injury site and accumulate at the border zone in the post-infarction adult mouse heart and it fails to regenerate.  While these data suggest that SNs play an important role in heart regeneration, when and how the SNs migrate into the injury site and their role(s) in heart regeneration remain unknown.

Here using the zebrafish heart, which regenerates post-injury, we have analyzed when and how SNs migrate into the injured tissue.  Using various reporter lines and antibodies, we find that the zebrafish SNs migrate rapidly into the injured tissue.  A subset of nerves persists within the injured tissue for several days and the innervation pattern is re-established within 30 days post-injury.  This rapid response of SNs is accompanied by the disintegration of the pre-existing neural network.  Altogether our studies indicate that after cardiac injury, cardiac SNs are very dynamic.  Hence, analyzing the function of SNs after cardiac injury will significantly advance our understanding of their role(s) during organ regeneration and cardiac disease.

Clinical and preclinical studies reported insufficient cell engraftment and delayed cardiac functional recovery problems in stem cell-based therapy in heart failure. Therefore, pretreatment of stem cells with an exogenous factor may improve regenerative efficiency. Here we demonstrated the effect of estrogen administration on the regenerative capacity of CPCs with in vitro and in vivo experiments. Explant-derived CPCs were isolated from adult mouse hearts and molecular, functional, and electrophysiological changes in CPCs upon estrogen treatment were investigated. Estrogen treatment increases the expression of proliferation, pluripotency and stem cell markers like Abcg2 and Islet-1. The migration and vasculogenic potential of CPCs was also induced by estrogen. Considering the paracrine effect of transplanted progenitor cells, we have examined the changes in the exosomal miRNA profile of CPCs. We found that estrogen regulates exosomal miRNAs known as modulators in neo-angiogenesis and cell migration. These results provided molecular evidence that estrogen pretreatment improves the regenerative capacity of CPCs inducing their self‐renewal, potency and repairment mechanisms. We have also monitored ohmic behaviour for K-channels like inward rectifier K+-current and significant hyperpolarization-shift in inward currents upon estrogen treatment suggesting the establishment of the resting membrane potential and differentiation induced by estrogen treatment. We have observed the inductive role of estrogen on cardiomyocyte differentiation of CPCs. To support in vitro data, we have used mouse heart failure model established by isoproterenol injection. Molecular and electrophysiological changes after the intracardiac allogenic transplantation of estrogen pretreated CPCs were also demonstrated. All these results indicate that ex vivo pretreatment of CPCs with estrogen may propagate the transplantation efficiency.

Cardiomyocyte proliferation is a critical source of new myocardium during heart development and regeneration. Novel therapies designed to replace missing or lost cardiomyocytes in the context of congenital heart disease (CHD) or myocardial infarction (MI) represents the holy grail of cardiac regenerative medicine. To boost cardiomyocyte proliferation in either setting, critical regulators must be identified. We reported that Reptin (also known as Ruvbl2), a highly conserved and widely expressed AAA+ ATPase, is a potent suppressor of cardiomyocyte proliferation during heart development and regeneration. Specifically, reptin loss-of-function mutants display ventricular hyperplasia at 3 days post-fertilization (dpf), while cardiomyocyte-specific reptin overexpression causes ventricular hypoplasia. Moreover, heat-inducible reptin overexpression during adulthood also lowers the myocardial proliferation index and results in scarring. Building on these published data, I am currently investigating whether the suppressive activity of Reptin on cardiomyocyte proliferation is conserved in mice and dissecting the downstream molecular mechanisms mediating this function. To address the former, I created cardiomyocyte-specific Reptin knock-out mice by crossing cTnt:Cre;Reptin^fl/+ X Reptin^fl/fl. Of 38 liveborn pups, zero mutant animals were recovered. I found that the conditional mutants die by E12.5, demonstrating a strict requirement for murine heart development. I am currently investigating cardiomyocyte proliferation in E10.5 and E11.5 conditional knock-out embryos and analyzing differentially expressed genes identified by RNA-seq from wild-type and reptin mutant cardiomyocytes in zebrafish. 

Cardiovascular diseases remain the leading cause of death worldwide, and few effective treatment options are available. Myocardial injury, such as myocardial infarction, causes irreversible damage of the heart muscle and its replacement by scar, leading to a chronic decrease in heart function. In contrast to humans, the injured zebrafish heart muscle regenerates efficiently through robust proliferation of myocardial cells. Thus, the zebrafish presents a beneficial vertebrate model for studying genetic programs behind cardiac regeneration, which may be present, albeit dormant, in the adult human heart.

To this end, we established a novel platform for studying heart regeneration after cardiomyocyte ablation in zebrafish larvae. The specific ablation of cardiomyocytes is achieved through a transgenic construct inducing the expression of nitroreductase, a bacterial enzyme, in a pool of ventricular cardiomyocytes. Subsequent treatment with antibiotics induces cell death specifically in nitroreductase-expressing cells. In combination with automated 3D heart imaging, this platform can be used for medium-throughput screening of genes and compounds with presumed effects on regeneration. Our results confirm that we induce a robust loss of the targeted cardiomyocytes, which are replaced through the proliferation of remaining cardiomyocytes within 4 days post injury. Our results further show that treatment with known anti-regenerative molecules causes a significant delay in regeneration kinetics, providing a proof of principle for this platform in identifying anti-regenerative effects of genes and drugs. Using this platform, we aim to discover therapeutic targets and drugs that will allow us to activate the dormant regenerative potential of the human heart.

Cardiac progenitor cells (CPC), whose regenerative capacity is reduced due to cardiac damage, play a role in heart failure that develops after heart damage. Regenerative capacity of multipotent CPCs is being evaluated by clinical phase-1 studies. Due to the low rates of inoculation and differentiation, treatment modalities based on CPC transplantation need to be improved. Our aim in this study is to show how estrogen replacement affects cardiac regeneration and differentiation capacity of CPCs in female ischemic animals. Recent studies  have shown that estrogen has an active role in cardiac regeneration both in functional and molecular levels. In addition, in another study by our group, we showed that the effects of estrogen on CPC differentiation have a positive effect on the regenerative capacity of CPCs. Evaluation of estrogen-related changes in the regenerative capacity of female ischemic CPCs and healthy CPCs is important in order to better understand the role of estrogen in cardiac regeneration. To investigate this, we examined changes in endothelial, fibroblastic, and cardiac differentiation capacities of CPCs derived from healthy and ischemic heart explants upon estrogen replacement. Our results showed that estrogen has a positive effect on these parameters. We also examined estrogen-dependent changes in EMT markers (Vimentin, Snail, Slug, CDH1, and CDH2) affecting the engraftment rate. Our in vitro results showed a shift towards mesenchymal characteristics upon estrogen treatment. Our results with this study are a step towards a better understanding of the effects of estrogen on the regenerative capacity of the hearts of healthy or ischemic females.

Cardiac progenitor cells (CPCs) are a diverse group of cells found in the adult heart that can develop into distinct cardiac lineages. CPC transplantation has poor stem cell engraftment and delayed cardiac functional recovery difficulties in stem cell-based heart failure treatment. Therefore, exogenous factor therapy of CPCs may boost regeneration efficiency. Estrogen is recognized to play a role in heart regeneration on both a functional and molecular level. In vitro experiments, we wanted to see how estrogen treatment affected Sca1+ regenerative ability. The Sca1+ CPC subpopulation was isolated exclusively from mouse cardiac explants. When compared to mRNA levels in heart tissue, the expression level of pluripotency markers in sorted Sca1+ cells is significantly higher. Estrogen also promotes colony formation and proliferation. In wound healing and angiogenesis experiments, estrogen therapy promoted migration and micro vascularization in Sca1+ cells. The expression of mesenchymal markers has also increased, implying that the outcomes are functional. We also looked for ohmic activity in K-channels, which was identical to the inward rectifier K+-current, as well as a considerable hyperpolarization shift in inward currents after estrogen therapy. These data show that estrogen therapy promotes the development of the resting membrane potential and differentiation. All of these findings suggest that preconditioning CPCs with estrogen ex vivo before transplantation may improve transplantation efficiency and could be employed as a therapy option in clinical trials.

 Surjya Narayan Dash,1* Suneeta Narumanchi,2* Jere Paavola,2 Sanni Perttunen,2 Hong Wang,1 Päivi Lakkisto,2,3 Ilkka Tikkanen,2,4* and  Sanna Lehtonen1*

¹Department of Pathology, University of Helsinki, Helsinki, Finland; ²Unit of Cardiovascular Research, Minerva Institute for Medical Research, Biomedicum Helsinki, Helsinki, Finland; ³Clinical Chemistry and Hematology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; and ⁴Abdominal Center, Nephrology, Helsinki University Hospital, Helsinki, Finland

 * equal contribution

Actin is the most abundant and conserved protein in eucaryotic cells and involved in various cellular functions from cell division and migration to muscle contraction. Actin and myosin generate contractile force in cardiac muscle, and mutations in these proteins may lead to heart failure. Septins are small GTPases that associate with actin filaments and play an important role in cytoskeleton organization, however, their role in cardiomyocyte organization and function is not completely understood. We found that septin 7 is expressed in both embryonic and adult zebrafish heart. Knockdown of sept7b, the zebrafish ortholog of human septin 7, reduced F-actin and α-cardiac actin expression in heart and caused disorganization of actin filaments. Electron microscopy revealed disorganization of heart myofibrils and partial detachment from Z-discs in sept7b knockdown larvae. Functional studies showed that the knockdown larvae had reduced ventricular dimensions, contractility and cardiac output. Additionally, depletion of sept7b reduced the expression of retinaldehyde dehydrogenase 2 (raldh2), which catalyzes synthesis of retinoic acid (RA), necessary for heart morphogenesis. Furthermore, we found that sept7b and RA signaling pathways converge to regulate cardiac function. Together, these results identify an essential role for sept7b in zebrafish heart development and function.

During embryonic heart development, epicardial cells residing within the outer layer of the heart undergo epithelial-mesenchymal transition (EMT) and migrate into the myocardium to support and stimulate organ growth and morphogenesis. Disruption of epicardial EMT results in aberrant heart formation and embryonic lethality. Despite being an essential process during development, the regulation of epicardial EMT is poorly understood.  Here we report EMT relative to the topography of the epicardial surface of the embryonic heart at high resolution using scanning electron microscopy (SEM). We identified high- and low-EMT regions within the mesothelial layer of the epicardium and an association of these regions with key components of the extracellular matrix (ECM).  The ECM basement membrane-associated proteoglycan agrin was found to localize in the epicardium in regions actively undergoing EMT. Deletion of agrin resulted in impaired EMT and compromised development of the epicardium, accompanied by down-regulation of the epicardial EMT regulator WT1. Agrin enhanced EMT in human embryonic stem cell-derived epicardial-like cells by decreasing b-catenin and promoting pFAK localization at focal adhesions. In addition, we observed that agrin promoted the aggregation of its receptor dystroglycan within the Golgi apparatus in murine epicardial cells. Loss of agrin resulted in dispersal of dystroglycan throughout the epicardial cells in embryos, disrupting basement membrane integrity and impairing EMT. Our results provide new insights into the role of the ECM in heart development and implicate agrin as a critical regulator of epicardial EMT.  

Proper functional remodeling of atrio-ventricular junction (AVJ) leaving a sole myocardial connection in the annulus fibrosus (His bundle) is crucial for the post-septated heart development. Presence of myocardial accessory pathways (APs) bypassing the annulus fibrosus can lead to the ventricular pre-excitation and could lead to severe tachyarrhythmias. The aim of our study was to functionally characterize remodeling of AVJ and diminution of myocardial atrio-ventricular connection throughout chick cardiogenesis. At the embryonic day (ED) 14, 16 and 18, we used optical mapping of retrogradely perfused heart to determine atrio-ventricular delay and localize the APs. Measurements were performed during normal conditions and after atrioventricular node conduction slowing by adenosine injection. Hematoxylin-Eosin /Alcian Blue and Picrosirius Red staining were used for morphological validation of the APs. We observed a decreased proportion of hearts with electrically active APs from 60 % at ED 14 to 50 % at ED 16 and 20 % ED 18, respectively. At ED 14, the majority of the APs were detected during normal conditions and were distributed all around the AVJ. However, the most of the APs were detected after adenosine perfusion only at 16 and 18 ED, and were mostly right-sided. This was accompanied by a trend to decrease in atrio-ventricular delay during the studied period.  Progression of AVJ fibrotization with APs disappearance was confirmed by histology. We provide a view on functional AVJ remodeling throughout chick development. This could help to elucidate spatiotemporal characteristic of proper AVJ isolation and understanding of arrhythmias associated with presence of APs.

Crucial for heart function is its partition into a left and right pump. Left-right asymmetry of cardiac morphogenesis begins with the rightward looping of the embryonic heart tube, which positions cardiac chambers relative to each other to establish the double blood flow. In humans, disturbances in this process have been associated with severe heart defects in the heterotaxy syndrome. We have previously demonstrated that the left signal Nodal is required in cardiac precursors for orienting and shaping the embryonic heart loop (Desgrange et al., 2020). Nodal mutants later develop an heterotaxy syndrome with a spectrum of heart defects. However, the heterogeneity of the heterotaxy phenotype has remained poorly understood. Since Nodal mutants at E9.5 display four categories of abnormal heart loops, we reasoned that heterotaxy may be stratified. For example, in the clinical nomenclature, the final position of the ventricles is considered to reflect the direction of the embryonic heart loop. However, this correlation has never been tested experimentally. To tackle the challenge of monitoring a specific phenotype at two stages of development in a single embryo, we developed a multimodality imaging pipeline, combined with advanced 3D image analyses. With this unique approach, we can identify heart loop features that are predictive of specific cardiac malformations. This study provides novel insights into the emergence of complex congenital defects in the heterotaxy syndrome.

Pediatric and adult patients with Chronic Atrial and Intestinal Dysrhythmia (CAID) have a recessive point mutation (A > G) in the first coding exon of the cohesin interacting protein Shugoshin 1 (SGO1). This leads to a lysine-to-glutamic acid change at the highly conserved amino acid 23 in SGO1 (p.Lys23Glut: SGO1-K23E). To investigate the role of Sgo1 in vivo, we generated Sgo1-K23E and Sgo1-flox mouse models. Affected Sgo1-K23E/K23E homozygous mouse mutants die embryonically and show global developmental delay, consistent with an early embryonic requirement for Sgo1. Non-affected Sgo1-K23E/K23E homozygous mouse mutants survive postnatally at reduced Mendelian ratios and in a genetic background-dependent manner, but develop normally with no overt signs of functional or structural defects. Likewise, early postnatal conditional ‘knock-in’ of Sgo1-K23E similarly does not affect cardiac or intestinal function, but, surprisingly, leads to lack of neonatal hair growth. Mesodermal, second heart field, and neural crest lineage-specific Sgo1 knock-out mutants show complete embryonic lethality and lineage-specific defects shortly upon activation of Cre expression. Together, our results indicate that mouse Sgo1-K23E induced defects are genetic background dependent and that Sgo1 is likely globally required throughout development.

The genetic control of heart development has been extensively studied, partly due to the prevalence of congenital heart disease (CHD). CHDs primarily arise from mutations affecting gene dosage or altering spatial/temporal expression patterns, making non-coding elements that control these aspects of gene expression particularly important. However, the annotation and functional dissection of non-coding elements is lacking when compared to the coding genome.

Recently, our lab identified 8866 human regulatory elements deeply conserved between zebrafish and humans. Dubbed accessible conserved non-coding elements (aCNEs), these open chromatin regions are enriched for enhancers and are conserved at the DNA sequence level between zebrafish and humans. Due to the deep conservation of these regions, we hypothesize that a subset of aCNEs are conserved critical regulators of cardiac development and implicated in cardiac disease.

To identify regions with a likely role in cardiac development, I used publicly available ChromHMM data from human fetal hearts to identify aCNEs with chromatin signatures representing enhancer states. I then used published ChIPseq datasets to identify regions bound by known cardiac transcription factors (TF), including NKX2.5, GATA4/5/6, and TBX5. These criteria identified 329 regions, capturing previously identified cardiac enhancers, and predicting 258 novel cardiac enhancers.

Functional examination of 17 of these novel regions showed that 88% were able to drive reporter gene expression in the developing zebrafish heart. Strikingly, two of these regions contain ultra-rare SNVs in patients with familial Left-sided cardiac legions. Currently, we are working on linking upstream TFs and downstream target genes to aCNEs to gain molecular insight into how these regions function in cardiac development and how alterations may contribute to CHDs. Altogether, this work identified putative cardiac enhancers crucial in cardiac development and disease.

Congenital heart defects (CHDs) are the most common human birth defect, occurring in 1% of newborns. Cardiac neural crest cells (cNCCs) are a migratory and multipotent cell population known to aid in the development of the cardiac outflow tract (OFT), valves, and interventricular septum. We aim to establish the contribution of Yap and Taz effectors of the fundamental Hippo signaling pathway in neural crest (NC)-derived cardiac development. Conditional knockout (CKO) mice were created using a cre-lox system, by Wnt1^cre and Wnt1^cre2SOR, NC-specific drivers.Yap^+/-;Taz^-/- CKO mutants produced various OFT and remodeling defects including ventricular septal defect (VSD), tetralogy of Fallot, double outlet right ventricle, and cardiac valve aberrations, varyingly between E14.5, E16.5, and E18.5. Interestingly, Yap^+/-;Taz^+/- double-heterozygous hearts exhibited external morphology similar to that of controls, but sectioning revealed mild VSD, along with cardiac valve leaflet irregularities, at E16.5 and E18.5. Cell apoptosis and proliferation were unchanged in Yap^+/-;Taz^-/- CKO mutants. Compelling preliminary data utilizing various methodologies including RNA-sequencing, Reverse Phase Protein Array (RPPA), Assay for Transposase-Accessible Chromatin-sequencing (ATAC-seq), and transwell migration assay, indicate a potential role for Yap/Taz in regulating NC migration. Preliminary migratory studies are followed by both OFT culture and live-imaging. Together, our data indicate that Yap/Taz play a critical role in proper cNCC-derived cardiac formation.

Cornelia de Lange syndrome (CdLS) is a rare genetic and developmental disorder affecting about 1:10 000/1:30:000 children. Cardiac defects are observed in more than 50% of patients. CdLS children feature septal defects, and outflow tract defects including the hypoplastic aorta, stenosis, or coarctation of great arteries as well as Tetralogy of Fallot. We found that Nipbl+/- mice on a C57Bl/6 genetic background featuring a significant decrease in Nipbl mRNAs as well as a decrease in the protein in the heart also featured a severe delay in embryonic and postnatal growth. High-Resolution Episcopic Microscopy analysis of the heart at birth revealed ventricular hypertrophy, ventricular septation defects associated with a persistent truncus arteriosus. The adult hearts then feature a severe aortic phenotype with an enlargement of the intima including senescent cells and stenosis resulting in an increase in aortic flux velocity and persistent left ventricular hypertrophy. The aortic phenotype was recapitulated when Nipbl was deleted in smooth muscle cells using Sma^CreERT2 x Nipbl^fl/fl mice. Using proteomics and RNA-sequencing of the outflow tract, we identified a dysregulated TGFb and retinoic acid pathways in embryonic Nipbl+/- hearts as well as the presence of P21+  and pERK+ senescent cells as early as in E13.5 Nipbl+/- embryonic hearts.

Human CdLS patient iPS cells differentiated in smooth muscle cells also featured a severe senescence phenotype. The phenotype was reversed by an ALK5 inhibitor

Treatment of pregnant mice with an ALK5 inhibitor from E9.5 to E13.5 also rescued the cardiac phenotype as well as the body size of mice at birth. 

Down Syndrome (DS), caused by trisomy of human chromosome 21 (Hsa21), results in a range of phenotypes, including congenital heart defects (CHD). These phenotypes arise from increased dosage of one or more of the ~230 genes on Hsa21, however the gene(s) needed in three copies to cause CHD and the underlying mechanisms remain unknown.

The lab has generated mouse model for DS (Dp1Tyb) carrying a duplication of 23Mb on Mmu16, orthologous to Hsa21. These mice show the types of CHD seen in babies, such as ventricular septal defects and atrioventricular septal defects. The overall aim is to identify causative genes and understand the pathological mechanisms underlying CHD in DS.

Using our mapping-panel, we found that a critical gene for the CHD phenotype is Dyrk1a. Our data show that DS fetal hearts have decreased oxygen consumption and proliferation, suggesting that this is an important cause of the developmental defects. We show that three copies of Dyrk1a are required for CHD in Dp1Tyb. Three copies of Dyrk1a are also required for the mitochondrial and proliferation defects in embryonic cardiomyocytes.

Our preliminary analysis of the atrioventricular cushion (AVC) shows a reduction of cellular density as well as a reduction of NFATC1 signaling pathway, involved in valve morphogenesis. Thus, decreased NFAT signaling in the AVC might lead to decreased mesenchymal proliferation and cause the reduced AVC density in Dp1Tyb mice.

Taken together, we propose that CHD in DS arise in part from increased DYRK1A activity in cardiomyocytes leading to mitochondrial and proliferation dysfunction.

The epicardium is a layer of epithelial cells covering the surface of the heart that provides important cellular contributions for embryonic heart formation. In addition, seminal works during the last decade have pointed out that the adult epicardium is re-activated after heart damage contributing to cardiac remodeling. Several fate mapping and cell lineage studies have demonstrated that coronary vascular smooth muscle cells (cVSMC) and cardiac fibroblasts develop from the EPDCs in a multi-step process involving cell proliferation, epithelial-to-mesenchymal transition (EMT) and cell migration. However, the molecular signalling cascades regulating EPDC specification and migration are poorly understand. Here we show that miR-200b is expressed from E12.5 to E15.5 during heart development. FISH in situ hybridization analysis in Wt1Cre‐YFP, embryos, as well as, qPCR of sorted cells, showed that miR-200b is present in a cell subpopulation of EPDCs at these stages of cardiogenesis. RNA-pull-down together with RNAseq analyses reveal that all miR-200b-targets in epicardial cells are related to cell migration. In vitro experiments of gain and loss of functions, by using EPDCs and whole-organ cultures from mouse embryos, evidenced that miR-200b modifies cell motility. Additionally, analysis in ventricles from infarcted adult mice, strongly demonstrate that miR-200b is upregulated from post-myocardial infarction day 3, peaking at day 7. Collectively, our data suggest that this miRNA might be a key molecule regulating epicardial cell lineage migration during cardiac development, as well as, in EPDC activation after myocardial infarction.

A role for cardiac sympathetic hyperinnervation in arrhythmogenesis after myocardial infarction (MI) has increasingly been recognized. In humans and mice, the heart receives cervical as well as thoracic sympathetic contributions. In mice, superior cervical ganglia (SCG) have been shown to contribute significantly to myocardial sympathetic innervation of the left ventricular anterior wall. Of interest, the SCG is situated adjacent to the carotid body (CB), a small organ involved in oxygen and metabolic sensing. We investigated the remodeling of murine SCG, as well as the proximate CB, over time after MI. Murine SCGs were isolated from control mice, as well as 24 hours, 3 days, 7 days and 6 weeks after MI. SCGs were stained for autonomic nervous system markers (Tubb3, TH and ChAT), as well as for neurotrophic factors (BDNF and NGF) and their receptors. Quantification of the staining-intensity as well as the neuronal size was performed in the entire SCG and in the proximate CB. Our results show that ChAT and TH are co-expressed in SCG neuronal cells in control ganglia. After MI, neuronal remodelling occurs, with a significant increase in size of ganglionic cells and a decreased intensity of ChAT expression. This SCG remodeling was observed as early as 24 hours after infarction, with a peak at day 7, regressing within 6 weeks post-MI to basal levels. Of note, the most robust neuronal remodeling was observed at the region adjacent to the CB. An increase of neurotrophic factors (BDNF and NGF) was observed in the CB and neuronal cells, whereas the high affinity receptors for BDNF and NGF increased in the SCG after MI. These findings were concomitant with an increase in GAP43 expression indicating axonal outgrowth in the SCG. In conclusion, overt neuronal remodeling occurs after MI in the SCG as well as in the CB, suggesting an interaction of these 2 structures that might contribute to pathological cardiac hyperinnervation

During cardiac development the ventricle undergoes trabeculation, where a subset of cardiomyocytes delaminate from the myocardial wall and project into the heart lumen. At the onset of trabeculation the ventricular wall exhibits apicobasal polarity, where the exterior surface is apical and trabecular cardiomyocytes emerge basally. Despite recent advances, the mechanisms by which ventricular wall integrity is maintained as cardiomyocytes delaminate are still poorly understood.

Laminins are extracellular matrix proteins that are typically basally deposited, forming the basement membrane. We identified a dynamic basal-to-apical shift in laminin deposition in the developing ventricle: prior to trabeculation laminin localises to the luminal (basal) ventricular surface, but concomitant with initiation of trabeculation basal laminin is removed and instead deposited at the apical exterior of the ventricle. Analysis of the ventricular wall in laminin beta1a zebrafish mutants reveals an increased number of cardiomyocytes undergoing aberrant apical extrusion, suggesting apical laminin helps maintain myocardial wall integrity.

Llgl1 (Lethal(2) giant larvae protein homolog 1) is a component of the Scribble complex, which is important for maintaining apicobasal polarity including regulating Crumbs, an apical transmembrane protein required for ventricular wall organisation and trabeculation. We therefore hypothesised Llgl1 plays a role in ventricular wall polarisation, organisation, or trabeculation. Analysis of llgl1 zebrafish mutants revealed a delayed basal-apical laminin shift, alongside defects in Crumbs relocalisation. These defects are accompanied by an increase in ventricular cardiomyocytes undergoing apical extrusion. Together our data indicate that llgl1 maintains ventricular wall integrity during trabeculation through promoting timely establishment of an apical laminin sheath around the ventricle.

The mammalian adult heart is a post-mitotic organ characterized mainly by polyploid cardiomyocytes (CMs). The post-mitotic status of adult CMs poses a clear limit to heart regeneration. Recent studies have revealed that the PIDDosome, a multi-protein complex consisting of Pidd1, Raidd, and caspase-2, controls p53 activation in response to supernumerary centrosome and limits scheduled as well as accidental polyploidization events. Since PIDDosome function in heart remains elusive, the overall goal of this project is to unveil the role of the PIDDosome in CM polyploidization, as barrier to tissue regeneration.

Flow cytometry-based analyses of adult CM nuclei showed that the absence of either Pidd1, Raidd, or caspase-2 results in an increase of tetraploid CM nuclei. Moreover, cardiac-specific deletion of caspase-2 resembles the ploidy phenotype observed in Casp2 knockout mice, suggesting that the increased CM ploidy is a cell autonomous process. Comparative analyses between different postnatal stages highlighted a gradual increase in tetraploid CM nuclei in hearts from Pidd1, Raidd or Casp2 knockout mice compared to wild-type mice at postnatal day 7 narrowing down the time of PIDDosome action. Previous studies have demonstrated that Pidd1 must be anchored to mother centriole via Ankrd26 in order to activate the PIDDosome pathway. Preliminary data from Ankrd26 knockout mice revealed that similar to Pidd1, Raidd and Casp2  knockout mice, the absence of Ankrd26 leads to an increase in tetraploid CM nuclei. On the other hand, in p53 knockout mice the ploidy is not statistically increased compared to wild-type animals, suggesting that the PIDDosome regulates CM ploidy in a p53-indipendent way.

The atria serve as a contractile blood reservoir of the cardiac pump. During embryogenesis, the atria and ventricles are soon distinguished from the persisting tubular segments (atrioventricular canal, outflow tract) by disappearance of the cardiac jelly and formation of the pectinate muscles and trabeculae, respectively. Unlike the well-characterized and frequently investigated ventricular myoarchitecture, the arrangement of muscular bundles in the atria is insufficiently studied. Here we present the effects of experimental hemodynamic alterations on embryonic and adult structural and electrical properties of the atria in a variety of animal models. Chick embryonic left atrial ligation (LAL) led to inhibition of pectinate muscle development in the excluded portion of the left auricle and appearance of ectopic pacemaking activity in that region. In rat volume overload model induced by aorto-caval fistula (ACF), the atrial weights were significantly increased (similar to ventricles), with more pronounced response in the right atrium; this increase was significantly attenuated by renal denervation. In another experimental rat model of pulmonary hypertension (SUGEN-hypoxia), an extreme rise in right atrial and ventricular pressures was observed, with accompanying profound structural remodeling of both ventricles as well as the atria. Analysis of these models will provide us with clues regarding the mechanisms governing the normal development of atrial form and function and their perturbations in overload-induced remodeling and arrhythmogenesis.

Muscular dystrophies (MD) are a group of diseases that cause progressive weakness and loss of muscle mass. Mutations in components of the dystrophin-glycoprotein complex (DGC) cause several types of MDs, such as Duchenne. There are no effective cures available for MD and current treatments only modestly alter disease progression. This is mostly attributed to the lack of understanding of the precise causal mechanisms. While both cardiac and skeletal muscles share many functional and molecular similarities, DGC function in both tissues is not identical. Thus, in both animal models and human patients, skeletal muscle dysfunction develops much faster than abnormalities in the heart. To better understand cardiac involvement in MD, we used a mouse model of cardiomyocyte-specific knockout (KO) of Dystroglycan- a main component of the DGC. We evaluated cardiac function and molecular changes in mice during homeostasis at different ages, and during acute injury settings. We show that KO of Dystroglycan in cardiomyocytes leads to a broader loss of other DGC proteins. However, normal cardiac function in mutant mice is not affected until the age of six months, after which it is drastically impaired. Surprisingly, a panel of acute heart injuries in young mutant mice do not reveal exacerbated damage as compared to WT. Yet, when stressing young mutant mice by β-adrenergic receptor stimulation we observe a marked deterioration in heart function. Late induction of Dystroglycan KO in both young and adult mice causes a temporary impairment in cardiac function, which returns to normal within a few weeks. Taken together we show that, as in the clinic, mice lacking the DGC can function quite well (if not stressed) as young adults suggesting the existence of compensatory mechanisms to cope with the loss of DGC in cardiomyocytes. Understanding these compensatory mechanisms might reveal novel targets for protection in both cardiac and skeletal muscle tissues affected in MDs.

Branchiomeric head and neck muscles have a shared origin with second heart field cardiac progenitor cells in cardiopharyngeal mesoderm (CPM). Clonal analysis has identified a series of common skeletal and cardiac muscle lineages along the anterior-posterior axis of the developing pharynx. Progenitor cells giving rise to jaw opening muscles and the right ventricle, or facial expression muscles and the cardiac outflow tract, are found in anterior CPM. Posterior CPM gives rise to neck muscles, including the trapezius, and both arterial and venous pole myocardium. The retinoic acid (RA) signalling pathway is required for normal deployment of cardiac progenitor cells in posterior CPM and blocking RA signaling during a defined early time window leads to conotruncal and atrial septation defects. Here we show using pharmacological approaches that blocking RA signaling also results in a strikingly selective loss of the trapezius muscle, without affecting other branchiomeric or somite-derived trunk and limb muscles. This reveals that, although regulators such as the transcription factor and 22q11.2 deletion syndrome gene TBX1 are broadly required for branchiomeric myogenesis, regulatory subprograms differ in posterior and anterior CPM. RA is required to specify the trapezius anlagen upstream of expression of myogenic determination factors of the MyoD family. Lineage specific activation of a dominant negative RA receptor suggests that, unexpectedly, this effect is not mediated by direct RA signaling to trapezius progenitor cells in CPM, but indirectly through the somitic lineage. These findings suggest a model in which trapezius development is dependent on a signaling cascade between cell types across the head/trunk interface. Our results provide insights into the mechanisms driving myogenic development within CPM and contribute to a better understanding of muscle pathology and evolution.

During development, the heart growths through addition of progenitor cells to the poles of the primordial heart tube.  In the zebrafish, wilms tumor 1 transcription factor a (wt1a) and b (wt1b) are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a/b expression is maintained in proepicaridal cells, it is downregulated in those pericardial cells contributing to cardiomyocytes from the developing heart. Sustained wt1 expression impaired cardiomyocyte maturation by reducing chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a/b-expressing cardiomyocytes changed their cell adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1 acts as a break for cardiomyocyte differentiation and ectopic wt1 expression in cardiomyocytes can lead to their transdifferentiation into epicardium.

Haploinsufficiency of TAB2 causes Congenital Heart Disease (CHD). TAB2 mediates non-canonical TGF-β signalling via interactions with TAK1 and TRAF6. Mutations of TAK1 cause Frontometaphyseal Dysplasia (FMD) and cardiospondylcarpofacial syndrome (CSCFS), rare multisystem syndromes, where CHD may appear in the clinical spectrum.

Whole exome sequencing data from a cohort of 1,471 syndromic CHD patients (sCHD), 2,405 patients with isolated CHD (iCHD) and 45,082 controls show increased burden of rare TAB2 and TAK1 variants in sCHD, but not in iCHD. We hypothesized that TAB2 and TAK1 are functionally important in heart development and addressed this experimentally in vitro and in vivo. Using in vitro models and embryonic tissues, we show that TAB2 and TAK1 are expressed in embryonic hearts and localise to primary cilia. Furthermore, mutation of Tak1 and Tab2 inhibits cardiomyogenesis of P19CL6 stem cells. In vivo zebrafish models show that mutation of tab2 and tak1 cause cardiac developmental defects. In addition, deep phenotyping of tak1 mutant zebrafish showed that several phenotypic features of FMD and CSCFS appear in the zebrafish model during early stages of development. This suggests a potential novel in vivo model for investigating FMD and CSCFS. In summary, our data suggests an important role for TAB2 and TAK1 in cardiac development and CHD, coordinated via the primary cilium.

　　　　　A subset of endocardial cells is hemogenic during early embryogenesis. Hemogenic endocardial cells are enriched in the cushion region and undergo endocardial-hematopoietic transition via Nkx2-5-dependent manner, suggesting that Drosophila tinman-dependent cardio-hematopoietic program is conserved in mammals. These hemogenic endocardial cells give rise to a subset of cardiac tissue macrophages that are essential for the cushion remodeling.

　　　　　To examine the regulatory network of Nkx2-5-dependent endocardial hematopoiesis, we analyzed scRNA-seq data from wildtype and Nkx2-5-null embryonic hearts. As expected, Nkx2-5-null hearts were devoid of clusters for hemogenic endocardium (e.g. Runx1⁺, Cd41⁺) and cushion endocardium (e.g. Twist1⁺, Msx1⁺). Interestingly, scRNA-seq analysis further revealed that genes related to Notch signaling pathway are significantly downregulated in Nkx2-5-null endocardium. To examine whether Notch signaling induces Nkx2-5-dependent endocardial hematopoiesis, we performed genetics experiments. Nkx2-5-null endocardium showed significant downregulation of Notch1⁺ endocardial cells, and NICD overexpression drastically activated hematopoiesis in the endocardium. Interestingly, impaired hematopoiesis and cushion defect in the Nkx2-5-null heart were both rescued by overexpression of NICD. Further gene regulatory network analysis identified that Dhrs3, which is known to catalyze the reduction of all-trans-retinaldehyde to all-trans-retinol and attenuates retinoic acid signaling, was a signature gene of the hemogenic endocardial cells downstream of Nkx2-5. Forced activation of NICD increased the number of not only Dhrs3⁺ hemogenic endocardial cells but also Dhrs3⁺ macrophages, suggesting a possible involvement of Dhrs3⁺ macrophages in cushion remodeling.

　　　　　This study demonstrated that the Nkx2-5/Notch signaling axis plays a pivotal role in endocardial-hematopoietic transition during early embryogenesis, thereby facilitating local tissue remodeling.

Most cases of congenital heart disease (CHD) occur sporadically and are, in part, attributable to rare de novo variants in chromatin regulatory genes, shown to account for ~3% of sporadic CHD by the Pediatric Cardiac Genomics Consortium (PCGC). This increases to 20% in cases of CHD that co-occur with neurodevelopmental disabilities (NDDs) and/or extra-cardiac anomalies. We analyzed the exome from whole genome sequence in 456 cases with conotruncal heart defects (CTDs) versus 537 controls with no heart anomalies, all with 22q11.2 deletion syndrome (22q11.2DS), to identify genetic modifiers. 22q11.2DS is ideal for this study as it has variable phenotypic expressivity of CHD and NDDs, along with other congenital abnormalities. Among the most interesting genes found was a subset of chromatin regulators that account for 9.21% of the CTD patients. We cross-referenced our list of 40 chromatin regulators with 90 identified in sporadic CHD by the PCGC, finding significant overlap (n=12; p<3.16e^-4) between the two groups. This is particularly compelling when considering that the identification of these variants was performed using different patient cohorts and statistical methods. Further, the chromatin regulatory genes in our cohort are overwhelmingly associated with genetic syndromes with increased incidence of CHD, NDDs, and extra-cardiac anomalies, making it of interest to determine whether they may also serve as modifiers for other syndromic anomalies. Thus, the data from our 22q11.2DS cohort reinforces the importance of chromatin regulatory genes as risk factors for sporadic CHD with syndromic features and suggests that modifiers of 22q11.2DS have inter-syndromic significance.

The cardiovascular system, comprised of the heart and the blood vessels, distributes oxygen and nutrients to the body. Accordingly, defective cardiovascular development can cause congenital heart disease (CHD). We have previously elucidated the mechanisms by which the transmembrane protein NRP1 promotes the remodelling of the foetal cardiac outflow tract (OFT) into the base of the pulmonary artery and aorta. NRP1 also enables the remodelling of the primitive pharyngeal arch arteries (PAA) into the great vessels that distribute blood from the heart into the lungs and body. We are seeking to (a) define the temporal window in which NRP1 enables PAA morphogenesis, (b) elucidate which specific cell types require NRP1 for PAA morphogenesis and (c) identify the NRP1 ligand(s) that mediate PAA morphogenesis. Here we show that NRP1 acts in both endothelial cells from the second heart field to promote PAA formation and in the cardiac neural crest lineage to promote vascular smooth muscle cell differentiation on the PAA, thereby promoting two consecutive stages of PAA morphogenesis. We have also begun to compare the requirement of different NRP1 ligands for these consecutive stages of great vessel formation. Defining the molecular and cellular mechanisms by which NRP1 enables PAA morphogenesis will increase our understanding of the normal and abnormal development of the cardiovasculature. In the long run, this knowledge may help improve the early diagnosis and treatment of CHD that involve the aortic arch arteries.

Wt1 encodes a zinc finger protein whose best-known function is its role as a transcription factor. Although for many years WT1 has been considered as one of the main hallmarks of the embryonic epicardium, new pieces of evidence have demonstrated the expression of WT1 in coronary endothelial cells during the vascularization of the embryonic heart and following myocardial infarction. Here we have used Wt1 reporter and lineage tracing mouse models to fully characterize the expression of WT1 in the endothelium through all developmental stages of coronary formation. To investigate its role during coronary vessel formation we have generated an inducible endothelial-specific Wt1 KO mouse model (Wt1-KO^ΔEC). The analysis of these mutant mice has demonstrated that WT1 expression in coronary endothelial cells during coronary plexus formation is required for the correct development of the coronaries and the compact myocardium. The transcriptomic analysis of coronary endothelial cells from Wt1-KO^ΔEC mice demonstrates that WT1 works as a master regulator of crucial processes of endothelial cells. We next used the postnatal retina, to investigate whether Wt1 functions in endothelial cells are conserved among other vascular beds. We observed profound alterations in the retinal endothelial network of postnatal Wt1-KO^ΔEC mice, revealing a reduced endothelial migratory phenotype and severely reduced vascular complexity. Our results shed new light on the functions of WT1 in the endothelium and suggest interesting roles for WT1 during physiological blood vessel formation. We speculate that pharmacologic interventions of WT1 or WT1 downstream targets may be useful for stimulating coronary vascular growth.

Heart development begins with the formation of a tube, as contralateral cardiac progenitors migrate and merge medially. Defective cell movement results in improper heart tube formation and congenital heart defects. However, the mechanisms of cell migration in early cardiogenesis remain unclear. In Drosophila, the embryonic heart is a linear structure composed of 52 pairs of bilateral cardiac precursors (cardioblasts, CB), that migrate dorsally and medially to form the heart tube. Using quantitative time-lapse microscopy, we found that CBs took periodic forward and backward steps as they migrated. The forward steps were greater in both amplitude and duration, resulting in net forward movement. Force generation by the molecular motor non-muscle myosin II is critical for cell movements. We found that myosin displayed an alternating pattern of localization between the leading and trailing ends of migrating CBs, forming oscillatory waves that traversed the cells. Live imaging revealed that the alternating myosin polarity was associated with alternating contractions of the leading and trailing edges. Mathematical modeling predicted that forward migration requires the presence of a boundary at the trailing edge of CBs that restricts backward movement. Consistent with this, we found a supracellular actin cable at the trailing edge of the CBs. Releasing the tension sustained by the supracellular actin cable increased the amplitude of the backward steps of CBs, thus reducing the speed of migration. Our results indicate that periodic cell shape changes coupled with a supracellular actin cable result in asymmetrical forces that guide cardioblast migration.

Congenital heart disease (CHD) remains the largest cause of birth defects worldwide, affecting about 1% of live births. Human genetics studies have identified genes potentially implicated in CHD, but the function of many of these genes in heart development and the specific cell types affected by their perturbation remain unknown. Furthermore, recent bulk and single-cell omics approaches have extensively characterized the molecular landscape of heart development, also identifying candidates expressed at various developmental stages. However, the functional relevance of many of these candidates remains largely unknown. The enormous search space for functionally important players in heart development and disease collectively created by these studies is challenging to approach with individual functional studies such as mouse knockout models. Here, we take advantage of the well-established system of hPSC differentiation to cardiac cell types, coupled with the recently established CRISPR-based perturbation method, CRISPR activation/inhibition, to perform a screen of 500 candidates potentially involved in the specification and differentiation of cardiac cell types. We will first establish our screen in atrial and ventricular cardiomyocytes, with the goal of expanding our approach to other cardiac cell types in the future. Taking advantage of the faithful recapitulation of key developmental stages in the hPSC system, we expect to uncover genes essential for the formation of differentiated cell types as well as their intermediary progenitor stages. Thus, by moving from expression to function at large scale, we hope to contribute to an enhanced understanding of cell-type specific molecular mechanisms of heart development and disease.

The Second Heart Field (SHF) is a population of cardiac progenitor cells that contributes to the developing heart after the initial formation of the primary heart tube. At the arterial pole, the SHF contributes to the outflow tract (OFT), the right ventricle (RV), and the ventricular septum (VS). At the venous pole, the SHF contributes to some atrial myocardial structures and to the dorsal mesenchymal protrusion (DMP). The importance of the SHF for normal heart development is demonstrated in studies in which perturbation of gene expression in the SHF has shown to result congenital heart malformations, including atrioventricular septal defects (AVSDs).

The AV mesenchymal complex (AVMC) consists of four separate entities; the two major endocardially-derived AV cushions, the mesenchymal cap situated on the leading edge of the developing primary atrial septum, and the DMP. Proper development of the AVMC is essential for the development of the septal structures at the AV junction

It is well-known that the transcription factor Sox9 plays an important role in the development of the AV cushions and valve formation. Preliminary studies and analysis of data in published papers by others, led us to believe that Sox9 could play a role in SHF-dependent development of the AV valvuloseptal complex. To test this hypothesis, we generated a SHF-specific Sox9 knockout mouse and studied how depleting Sox9 from this cell population affected the formation of the components of the AVMC and the impact of this experimental approach on AV septation.  The results of the study show that SHF-derived Sox9 plays a critical role in atrial as well as ventricular septation.

Pitx2 is recognized as a late effector of laterality regulating cardiac asymmetric remodeling, however the role of the gene in the context of cardiac differentiation and morphogenesis is still mostly unexplored. Pitx2 null mice present severe congenital heart diseases (CHD) including atrial and ventricular septal defects, and double outlet right ventricle. We planned to assess the dynamics of Pitx2 contribution to heart development and the link between absence of Pitx2 and CHD onset. To this aim, we combined morphogenetic and molecular analysis of Pitx2 mutant embryonic hearts with pseudolineage tracing of Pitx2^null cells. Here, we show that Pitx2 delineates a subpopulation of left cells which undergo directional expansion during cardiac development. This process drives concomitant ingression and progressive reallocation of the adjacent Pitx2^–ve population inside the heart. Directional expansion of the Pitx2 cell population progressively coordinates all aspects of external and internal remodeling through cardiac development, thereby directing ventricular sliding, outflow tract spiraling and septation. Our results provide a novel framework to understand the role of laterality in the developing heart and the rationale for congenital heart disease onset in laterality mutants.

A pathogenic variant p.G125R (c.373G>A) in the 5^th exon of TBX5 (T-box transcription factor 5) induces sick sinus syndrome and sinus bradycardia, indicating dysfunction of the leading pacemaker of the heart, the sinoatrial node (SAN). While homozygous mice die in utero, heterozygous mice with this variant (Tbx5^G125R/+) are morphologically unaffected but display longer and more variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, recapitulating the electrophysiological abnormalities seen in human carriers (van Ouwerkerk, 2022). Tbx5^G125R/+ mice present with a prolonged sinus node recovery time and sinus bradycardia, indicating SAN dysfunction. Tbx5-p.G125R clearly influences the atrial phenotype, however, its effects on the SAN are unclear. Here, we have found 3221 differentially expressed genes (P_adj <0.05) in the Tbx5^G125R/+ SAN; 1432 were downregulated and 1789 were upregulated. Several SAN-specific markers, key SAN transcription factors, and bone morphogenetic protein signaling components involved in SAN identity and development are differentially expressed in the Tbx5^G125R/+ SAN. Nevertheless, 3D reconstruction analyses of prenatal hearts indicated that SAN morphology is unaffected. However, fibroblasts are depleted in Tbx5^G125R/+ atria, and fibroblast- and extracellular matrix composition-associated gene expression is further decreased in Tbx5^G125R/+ SAN tissues, suggesting structural and compositional abnormalities. Altered expression of calcium handling genes and ion channels involved in normal SAN function further indicate that the Tbx5^G125R/+ SAN is functionally affected. We propose that Tbx5-p.G125R has a broad effect on the regulation of key drivers of SAN development, structure, cellular composition, and electrophysiological function.

GATA6 is a member of the GATA family of transcription factors that play an important role during heart development. GATA6 variants are primarily associated with congenital heart disease (CHD) and pancreatic agenesis. However, the phenotypic spectrum has expanded, including additional congenital malformations affecting the biliary system, congenital diaphragmatic hernia and developmental delay. Here, we report a family where the father and child duo had persistent truncus arteriosus (PTA) and the proband had atrial septal defect (ASD). Notably, the father had childhood-onset diabetes mellitus and the affected child was diagnosed with spontaneous intestinal perforation (SIP) on day 2 after birth, which has not been previously associated with GATA6 mutation. Using exome sequencing, we identified a novel, heterozygous missense variant in GATA6 (c.1403G>A; p.Cys468Tyr) in affected members. The variant in our family is absent from the gnomAD and ExAC databases and causes a missense change predicted to be damaging by all in silico tools. Sanger sequencing confirmed the exome sequencing results and segregation of this variant. GATA6 p.Cys468Tyr mutation affects the highly conserved zinc-finger domain of GATA6 and impedes its ability to bind DNA. In vitro functional analysis showed that p.Cys468Tyr mutant protein exhibited significantly decreased transcriptional activity. Furthermore, p.Cys468Tyr mutant demonstrated an impaired localization pattern and protein aggregation. Our findings expanded the variant and phenotypic spectrum for GATA6. Our study highlights that GATA6 variants can cause not only cardiac and pancreatic malformations but also gastrointestinal abnormalities, including SIP. Further investigation is needed to define the mechanisms underlying the full phenotypic spectrum associated with pathogenic variation in GATA6.

During cardiac morphogenesis, the linear heart tube expands to create cardiac chambers, each with a convex outer curvature (OC) and a concave inner curvature (IC). This stereotypical chamber shape facilitates the function of the embryonic heart, and errors in chamber morphogenesis are frequently associated with congenital heart disease. Using zebrafish as a model organism, our studies show that regional changes in cardiomyocyte morphologies underlie curvature formation: as the ventricle emerges, OC cells enlarge and elongate along their lateral axis, whereas IC cells remain relatively small and round while extending along their apicobasal axis. These divergent growth behaviors result in more squamous cells in the OC and more cuboidal cells in the IC. Coupled with these changes in cell morphology, we find distinct organization of the actomyosin cytoskeleton in each curvature: whereas F-actin and phospho-Myosin are distributed primarily along the basal and lateral cortices of developing OC cells, IC cells exhibit relative enrichment of this network along their apical cortex. These data suggest a link between actomyosin dynamics and patterns of cell shape change; indeed, we find that modulation of actin polymerization or myosin activity disrupts curvature-specific cell shapes. We therefore hypothesize that ventricular curvature formation involves the coordination of curvature-specific reorganization of the actomyosin network with acquisition of squamous and cuboidal cell morphologies in the OC and IC. Intriguingly, we find that the T-box transcription factor Tbx5a is required for both of these processes. Ongoing studies aim to connect the effector genes downstream of Tbx5a with the regulation of actomyosin organization and the attainment of OC and IC cell morphologies. Altogether, our work provides a new model for the control of chamber morphogenesis by localized cytoskeletal dynamics that create patterns of cardiomyocyte cell shapes.

Background/Introduction

Tbx1 is the major developmental gene involved in 22q11.2 deletion syndrome (22q11.2DS), the most common known genetic cause of congenital heart disease (CHD). The clinical phenotype is well recapitulated in Tbx1 mouse mutants, including cardiovascular abnormalities affecting the aortic arch, ventricular septum and cardiac outflow tract (OFT).

Intriguingly, clinical studies have linked VEGFR3, a gene that is regulated by TBX1, to CHD; rare variants in VEGFR3 predispose to OFT malformations, including Tetralogy of Fallot (TOF), the most common CHD found in 22q11.2DS patients. We have shown that in mice, TBX1 regulates Vegfr3 in endothelial cells. Furthermore, these genes interact strongly during cardiac lymphangiogenesis ensuring the correct number, morphology and growth of cardiac lymphatic vessels. Thus, Tbx1-Vegfr3 compound mutants are, potentially, a good model for genetic studies to reveal the site (tissue) and timing of the critical interaction in OFT development. 

Purpose

We hypothesize that TBX1 and VEGFR3 play essential roles in endothelial-mesenchymal transition (EMT), a key process in OFT formation. To test this, we performed a histological analysis of hearts of conditional Vegfr3 homozygous (Tbx1^Cre/+; Vegfr3^flox/^flox) embryos at E18.5.

Results and Conclusions

The results shown that Tbx1-driven inactivation of Vegfr3 causes a variety of intracardiac anomalies, including morphogenesis defects of the OFT. Our preliminary analysis provides proof of a genetic interaction between Tbx1 and Vegfr3 in cardiac morphogenesis and confirms the importance of Vegfr3 for cardiac development in the mouse. Future studies, will use time- and tissue-specific gene mutation in order to refine our genetic study.

Allelic heterogeneity in titin truncating variants (TTNtv) causes variable phenotypes and is associated with dilated cardiomyopathy as well as atrial fibrillation. However, limited knowledge is available regarding the cellular and molecular mechanisms behind the various disease manifestations. In order to improve our understanding of the cardiac pathology in patients carrying TTNtv, we investigated two different zebrafish models carrying an I- and A-band TTNtv, respectively. Using electrocardiography and echocardiography, we characterized the phenotype of adult heterozygous carriers of the TTNtv. The I-band truncated mutant was primarily affected in ventricular structure and function, while the A-band truncated mutant had electrical disturbances in the atrium already from early adulthood. Therefore, we set out to investigate whether there were any signs of cardiac dysfunction at the larval stage. Using in vivo calcium imaging, we found atrial calcium mishandling in three-week-old TTNtv-A heterozygous mutants while TTNtv-I heterozygous mutants displayed no defects. Additional findings were present as early as 5 days-post-fertilization, including increased sarcomere length in TTNtv-A heterozygous mutants and decreased ERK signaling only in TTNtv-I heterozygous mutants. These data show that TTNtv have molecular and physiological implications that affect the cardiac chambers differently depending on the position of the mutation.

Nr2f transcription factors are conserved regulators of vertebrate atrial development, yet the mechanisms by which these proteins function within atrial cardiomyocytes (ACs) are still not completely understood. To investigate the consequences of Nr2f loss within the atrium, we performed transcriptomic analysis on isolated ACs from wild-type (WT) and nr2f1a mutant zebrafish at 48 hours post-fertilization (hpf). Interestingly, our results revealed altered expression of core genes, including increased tbx3a and decreased nkx2.5, which respectively promote and repress sinoatrial node (SAN) differentiation. Subsequent examination of nr2f1a mutant atria from 48 to 96 hpf showed a progressive expansion of SAN markers into the atrium and concurrent regression of Nkx2.5 from the venous pole, with analysis of heart rate and action potential duration suggesting that nr2f1a mutant ACs functionally adopt pacemaker identity. Genetic epistasis using a heat-shock inducible Nkx2.5-EGFP transgene revealed that overexpression of Nkx2.5 can repress the SAN expansion in nr2f1a mutant hearts. Furthermore, profiling of chromatin accessibility in isolated ACs identified a putative nkx2.5 enhancer harboring an Nr2f binding site, which we found is expressed in the ACs adjacent to the SAN in transgenic embryos. Expression of the putative enhancer is lost in nr2f1a mutants, further supporting that Nr2f1a may directly maintain a border of atrial nkx2.5 expression. Altogether, our results reveal a novel requirement for Nr2f transcription factors in maintaining AC identity at the expense of SAN identity by maintaining Nkx2.5 expression in vertebrate hearts, which may provide insight into the etiology of arrhythmias and CHDs associated with human NR2F2 mutations.

Cardiomyopathy is a unified term for heterogeneous, progressive diseases of the myocardium that result in an enlarged, thickened or stiffened pathophysiology which, ultimately, disrupts the normal function of the heart.

A homozygous pathogenic variant, p.Arg253Trp SLC5A6, has been identified in a family whereby two siblings presented with dilated cardiomyopathy. SLC5A6 encodes the Sodium-dependent Multivitamin Transporter, a transmembrane protein that is crucial for facilitating the active transport of three vitamins: biotin, pantothenic acid and lipoic acid; all of which are critical, organic enzyme cofactors required for energy metabolism in the mitochondria.

Energy metabolism is a fundamental process in cardiac maintenance and, as the most metabolically demanding organ in the body, the heart requires a high ATP turn over to achieve efficient contractile function of the myocardium. To date, there is little known about the expression of SLC5A6 in the heart therefore, transgenic mouse models have been generated and various immunohistochemical, RNA and protein techniques have been performed in order to investigate its role.

A conditional deletion of Slc5a6 in cardiomyocytes results in the development of cardiomyopathy in mice and electron microscopy of Slc5a6 cardiac-specific knockout mice hearts showed mitochondrial degradation.
It is thus predicted that defects in Slc5a6 will prevent the transport of mitochondrial enzyme cofactors and underpin the progression to cardiomyopathy. Therefore, this project not only aims to establish the role of SLC5A6 in the heart but to investigate whether vitamin supplementation to Slc5a6 cardiac-specific knockout mice will delay progression to cardiomyopathy.

The Cardiac Jelly (CJ) is a specialized extracellular matrix (ECM) found between the myocardial and endocardial layers in the embryonic heart.  Developmental processes such as cardiac looping and ballooning depend on remodeling and maturation of the CJ by small leucine-rich proteoglycans such as Proline Arginine Rich Ends Leucine-rich repeats Protein (Prelp).  Prelp interacts with large ECM proteins such as Laminins and Collagens and is hypothesized to act as an anchor between the Basal Lamina (BL) and Reticular Lamina (RL).  Using imaging of live and fixed samples, we found that zebrafish prelp is required for the assembly of the cardiac BL as assessed by Laminin localization.  Loss of prelp leads to an increased number of cardiomyocytes, enlarged atrium and weak cardiac contractility.  Myocardium-specific rescue of prelp expression rescues the atrial chamber morphology defect.  Injecting mRNA encoding amino-terminal regions of Prelp in prelp mutants partially rescues the BL Laminin localization defect and improves cardiac function.  Transcriptomic analysis of prelp mutant hearts revealed the downregulation of basement membrane components such as Tenascin-C (tnc) and Fibronectin leucine-rich transmembrane protein 1b (flrt1b) which upon mRNA injections into prelp mutants rescued the BL Laminin localization defects.  Altogether, these data show the importance of small leucine rich proteoglycans in the BL in modulating atrium morphogenesis and myocardial-ECM interactions during early cardiac development.

Insights into the functions of a transcription factor ZFP516 in embryonic development are limited, except for its role in brown fat formation and regulation of stemness in embryonic stem cells. Germline homozygous loss of Zfp516 leads to neonatal lethality in mice, however, the cause of this lethality is not currently known. Here we find a role for ZFP516 in mouse embryonic heart development. Zfp516 homozygous loss results in outflow tract abnormalities at E14.5 such as the overriding aorta and in pulmonary or aortic valve phenotypes with full penetrance. Other cardiac phenotypes such as atrioventricular septal defect, perimembranous or muscular ventricular septal defects, or dorsal mesenchymal protrusion absence were observed in at least half of the E14.5 Zfp516 -/- embryos. At a molecular level we detected an upregulation of genes important in neurogenesis and synaptogenesis in murine hearts of Zfp516 -/- embryos at E13.5 with Nrn1 showing 15 fold increase in mRNA expression that was localized to a compact myocardium. At a functional level echocardiogram assessment of E13.5 murine hearts showed bradycardia with signs of atrioventricular block in Zfp516 -/- embryos, suggesting that there might be deficiencies in the function of the cardiac conducting system a day before structural defects were detected. Moreover, an investigation of lineage-specific requirements for ZFP516 in normal heart development showed a role in both the Sox10-expressing migrating neural crest and the Isl1-expressing second heart field lineages. Finally, germline analysis of >400,000 humans revealed loss of function mutations of the human ortholog are under negative selection suggesting an important role of the gene in the human development.

The endocardium plays important roles in the development and function of the

vertebrate heart, however few molecular markers of this tissue have been identified

and little is known about what regulates its differentiation. We describe here the

Gt(SAGFF27C); Tg(4xUAS:egfp) line as a marker of endocardial development in

zebrafish. Transcriptomic comparison between endocardium and pan-endothelium

confirms molecular distinction between these populations and time-course analysis

suggests differentiation as early as 8 somites. To investigate what regulates

endocardial identity, we employed npas4l/cloche, etv2/etsrp and scl loss-of-function

models. Endocardial expression is lost in cloche mutants, significantly reduced in etv2

mutants and only modestly effected upon scl loss-of-function. Bmp signalling was

also examined: overactivation of Bmp signalling increased endocardial expression,

whilst Bmp inhibition decreased expression. Finally, epistasis experiments showed that overactivation of Bmp signalling was incapable of restoring endocardial

expression in etv2 mutants. By contrast, overexpression of either npas4l or etv2 was

sufficient to rescue endocardial expression upon Bmp inhibition. Together, these

results describe the differentiation of the endocardium, distinct from vasculature, and

place npas4l and etv2 downstream of Bmp signalling in regulating its differentiation.

Jianan Wang, Joyce C.K. Man, Karel van Duijvenboden, Fernanda M. Bosada, Bas J. Boukens, Arie O. Verkerk, Geert J. Boink, Vincent M. Christoffels and Phil Barnett

AmsterdamUMC, AMC Amsterdam, Department of Medical Biology, Amsterdam, The Netherlands.

Expression of NaV1.8 (Scn10a), in cardiomyocytes, has been a topic associated with a certain level of controversy; is it there or not? With ongoing improvements in RNAseq output quality and sequence depth, genes and other genetic elements transcribed at low levels can now be detected with greater confidence. This lead to our observation that a short transcript comprising the last 7 exons of Scn10a, was being expressed in various cardiomyocyte components of the conduction system, cells in which we have never observed expression of Scn10a, or as we came to realize, full-length Scn10a. Aside from offering an explanation as to the expression controversy, we also show that this short version of NaV1.8 (Scn10a-short), also retains the ability to mediate an increased sodium current in the presence of the well described cardiac sodium channel NaV1.5 (SCN5A). Further, we have previously described a genetic variant (rs6801957), in an enhancer linked to the SCN5A/SCN10A gene locus and known to be associated with slow conduction, predisposing to arrhythmia. We now show that modification of this enhancer leads to reduced cardiac Scn10a-short expression, reduced sodium current, atrial conduction slowing and arrhythmia. Expression of the presumed target of the enhancer, Scn5a, remained unchanged.

Down syndrome (DS), trisomy 21, is a gene dosage disorder which results in multiple phenotypes including congenital heart defects (CHD). The genes on human chromosome 21 required in three copies to cause CHD and the altered cellular mechanisms that cause this pathology are unknown. We show that human DS fetal hearts and embryonic hearts from mouse models of DS have common transcriptional and proteomic changes, with reduced expression of oxidative phosphorylation and cell proliferation genes correlating with CHD. Using systematic genetic mapping, we identify Dyrk1a as a causative gene required in three copies to cause CHD and show that increased DYRK1A results in decreased mitochondrial respiration and reduced cell proliferation in embryonic cardiomyocytes. Thus, increased dosage of DYRK1A impairs mitochondrial function and causes CHD in DS.

Background:

Acute prenatal alcohol exposure (PAE) in combination with Notch mutations in the second heart field causes highly penetrant congenital heart defects (CHD), particularly conotruncal defects. The mechanism of this interaction and potential prophylactic interventions against it remain unexplored.

Hypothesis:

We hypothesize that alcohol inhibits Notch signaling via the gamma-secretase complex and that prophylactic folic acid supplementation would prevent this dysregulation.  

Methods:

We studied the effects of acute PAE in vivo in our murine model (two injections of 3g/kg 30% ethanol on E6.5) on cardiac phenotype, Notch signaling, SHF viability, and Notch intracellular domain (NICD) and presenilin-1 localization. Experiments were repeated using C2C12 cells exposed for 24 hours to growth media supplemented with 100mM ethanol with and without folic acid or its metabolite s-adenosyl methionine (SAM).

Results:

Acute PAE in vivo and in vitro resulted in reduction and intracellular redistribution of presenilin-1. NICD was retained in the cell membrane, reducing downstream Notch signaling. Both folic acid and SAM supplementation prevented these molecular changes. Folic acid supplementation preserved SHF proliferation, increasing proliferation 64% compared to those on a standard diet (p<0.001), and reduced CHD incidence 78% (p<0.001). 

Conclusion:

Acute PAE reduces Notch signaling through disruption of the gamma-secretase complex, inhibiting nuclear translocation of NICD. This results in reduced SHF proliferation and aberrant outflow tract development. Folic acid supplementation prevented the deleterious effects of alcohol. Further study is required to decipher the molecular mechanism coupling PAE to disruption of gamma secretase complex and the impact of folic acid supplementation on this mechanism. 

We previously showed that neural crest cell (NCC)-derived Wnt5a is crucially required for planar cell polarity (PCP) signaling in second heart field (SHF) progenitors and appropriate cardiac outflow tract (OFT) alignment. While previous studies indicate that histone modifying enzyme protein arginine methyltransferase-1 (Prmt-1) regulates Wnt5a expression in cranial NCCs during palatogenesis, there is limited understanding of the regulatory mechanisms of Wnt5a during heart development. We examined the impact of cardiac NCC Prmt-1 on Wnt5a expression and outflow tract maturation in mice. We found that Prmt-1 is globally expressed in migratory and post-migratory NCCs throughout outflow tract morphogenesis. Conditional knockout of Prmt-1 in NCCs (Wnt1-Cre) resulted in fully penetrant cleft palate and double outlet right ventricle (DORV) phenotype at E14.5. Cardiac NCC migration into the OFT was preserved in Prmt-1 mutants. However, SHF progenitor cells demonstrated perturbed PCP signaling and failed to migrate into the OFT, phenocopying SHF defects previously described in NCC-Wnt5a mutants. Wnt5a transcript expression was downregulated in Prmt-1 mutant NCCs by RNAseq, RT-PCR, and in situ hybridization. 75% of Wnt1-Cre, Prmt-1^F/+, Wnt5a ^F/+ double heterozygous mice developed DORV at E14.5, establishing the genetic synergy of Prmt-1 and Wnt5a pathways in vivo. Concomitant overexpression of Wnt5a-v5 in NCCs was sufficient to rescue DORV phenotypes in 71% (n=5/7) of Wnt1-Cre, Prmt-1^F/F mice. Taken together, we show that Prmt-1 regulates Wnt5a expression in cardiac NCCs and is required for SHF PCP signaling and OFT alignment. This work defines a novel role for Prmt-1 in cardiac NCC and outflow tract biology.

CHD7 is the chromatin remodeler haploinsufficient in CHARGE syndrome. Mouse models of Chd7 haploinsufficiency have cardiovascular defects similar to 22q11.2DS, and TBX1 haploinsufficiency. We assessed the role of CHD7 in the cardiopharyngeal mesoderm (CPM) by conditional mutagenesis and identified genes and pathways dysregulated in these mutants at E9.5. In cKO embryos cardiomyocyte differentiation genes and anterior SHF (aSHF) markers were decreased, but pSHF markers were increased, while expression of Tbx1 was not changed. We performed genome-wide profiling of CHD7 binding in cardiac progenitor (CP) cells and identified differentially regulated genes with CHD7 binding sites. Combination of our transcriptomics with the CHD7 binding profile shows that CHD7 acts at enhancers regulating first and second heart field gene networks, as well as a well characterized Fgf10 enhancer controlling differential expression in left ventricle versus second heart field. Thus, CHD7 is an important modulator of the mesodermal cardiogenic gene network.

Dilated cardiomyopathies caused by mutations in LMNA, encoding nuclear Lamin A/C, are highly malignant and prevalent. How LMNA mutations cause cardiomyopathies remains unknown. We characterized cellular, molecular, and pathological evolution of a mouse model of LMNA-related cardiomyopathy and provide evidence for a model in which nuclear envelope rupture unleashes nuclear-localized proinflammatory signaling, as a candidate molecular mechanism underlying disease pathogenesis. We observed that cardiomyocyte-specific, tamoxifen-inducible deletion of Lmna in adult mice (Lmna^CMKO) caused extensive fibrosis, increased macrophages, reduced ejection fraction, and chamber dilation by 3 weeks after Lmna gene deletion. Lmna^CMKO­ cardiomyocytes exhibited localized rupture of the nuclear envelope 2 weeks prior to the development of fibrosis and reduction of ejection fraction. Nuclear envelope rupture in Lmna^CMKO was immediately followed by an extensive upregulation of pro-inflammatory gene expression programs. We found that HMGB1, a potent proinflammatory protein normally sequestered in the nucleus, was released from the ruptured nuclei in Lmna^CMKO cardiomyocytes. Cardiomyocyte-targeted transcriptome analysis indicated that Lmna^CMKO cardiomyocytes did not upregulate the proinflammatory transcriptional program, raising the possibility that HMGB1 was further released to the extracellular space to active innate immune response in non-cardiomyocytes. Remarkably, treatment of Lmna^CMKO mice with immunosuppressant Dexamethasone prevented dilated cardiomyopathy, indicating that myocardial inflammation is the major contributor to the development of Lmna-related cardiomyopathy. Future work will examine the hypothesis that extracellular HMGB1 triggers pathogenic sterile inflammation leading to dilated cardiomyopathies in Lmna^CMKO mice. In conclusion, we identified the nuclear rupture-induced proinflammatory signaling as a candidate mechanism underlying LMNA-related cardiomyopathies.

Embryonic hypoxia is important mechanism driving heart development. Hif1α is a subunit of hypoxia-inducible factor 1 (HIF-1), regulator of transcriptional responses to decreased O₂ availability. Islet1 expressing cells are found in secondary heart field, right ventricle, proximal parts of coronary arteries and among others also in the sympathetic chain.

Hif1α ^loxP/loxP female mice were crossed with Islet1-Cre, Hif1α ^loxP/+, Cx40:GFP homozygous males to generate Hif1α CKO embryos, heterozygous for the Cx40-GFP reporter to visualize coronary artery endothelium. Whole mount imunohistochemistry (TH-tyrosine hydroxylase) was used to visualize sympathetic innervation. Further analysis was performed by immunohistochemistry and microCT. 

Hif1α CKO ED17.5 hearts exhibited misconnected main coronary arteries: septal artery misconnections, variations in proximal parts of coronary arterial vasculature, and variations in branching. Venous part of the embryonic vasculature is also analyzed.

Superficial nervous plexus of the Hif1α CKO embryonic heart was less developed mainly around the base of the heart compared to controls. The alignment and patterning of the nervous and vascular plexus on ventricular surface will be further analyzed.  

 In Hif1α CKO hypoxia induced signalization altered sympathetic heart innervation. Coronary vasculature is also affected by the loss of hypoxia signalization in SHF-derived myocardium. Together Hif1α CKO exhibit disrupted patterning of coronary vasculature as well as heart innervation and its navigation along the vasculature.

Hypoxic signaling is an important developmental mechanism in cardiac nerves and coronary vasculature development. Understanding mechanisms underlaying the development of cardiac nerves and vessels can clarify the mechanisms behind sudden cardiac death in young and adult patients.  

The mammalian heart arises from various populations of Mesp1-expressing cardiovascular progenitors (CPs) that are specified during the early stages of gastrulation. Mesp1 is a transcription factor (TF) that acts as a master regulator of CP specification and differentiation. However, how Mesp1 regulates the chromatin landscape of nascent mesodermal cells to define the temporal and spatial patterning of the distinct populations of CPs remains unknown. Here, by combining ChIP-seq, RNA-seq and ATAC-seq during mouse pluripotent stem cell differentiation, we defined the dynamic remodelling of the chromatin landscape mediated by Mesp1. We identified different enhancers that are temporally regulated to erase the pluripotent state and specify the pools of CPs that mediate heart development. We identified Zic2 and Zic3 as essential cofactors that act with Mesp1 to regulate its TF activity at key mesodermal enhancers, thereby regulating the chromatin remodelling and gene expression associated with the specification of the different populations of CPs in vivo. Our study identifies the dynamics of the chromatin landscape and enhancer remodelling associated with temporal patterning of early mesodermal cells into the distinct populations of CPs that mediate heart development. 

Ligand-Receptor inference analysis identified LDL receptor related protein 1 (LRP1), an endocytic receptor involved in regulating vascular remodelling, differentiation and cell migration, as a major signalling receptor in the developing epicardium. Currently, very little is known about the role of LRP1 in epicardial formation and function. Using an inducible epicardial-specific Cre (WT1-CreERT2), LRP1 was deleted from E9.5 (LRP1 iKO). LRP1 iKO embryos showed gross morphological defects including oedema, haemorrhage, and aberrant arrangement of the myocardial wall. Histological phenotyping of LRP1 iKO showed epicardial and coronary vessel defects at E14.5, with impaired invasion of epicardium-derived cells and abnormal vascular structures within surface nodules. By E17.5, the coronary vasculature was incorrectly patterned in LRP1 iKO and epicardial-derived vascular smooth muscle cells were largely absent. Epicardial epithelial-to-mesenchymal transition (EMT) and migration were further investigated in E11.5 explants from constitutive epicardial LRP1 KO hearts (Tg(Gata5-Cre)-driven; LRP1 cKO). Compared with controls, LRP1 cKO epicardial cells displayed reduced migration, incomplete EMT and cytoskeletal defects. These data suggest that LRP1 is required for epicardial EMT, a necessary process for differentiation to mural cell derivatives. EMT is critically influenced by signalling pathways including TGF-B, PDGF-B, FGF, Wnt and Notch1, all of which can be diversely controlled by LRP1, either through co-receptor activation or receptor clearance, through relaying to signalling pathways, or by modification and turnover of extracellular matrix and heparan sulphate proteoglycans. Ongoing studies seek to define the precise mechanisms through which LRP1 impacts epicardial signalling, to further our understanding of EMT in heart development and regeneration.

Tight regulation of retinoic acid (RA) levels is critical for normal heart development in all vertebrates. In zebrafish, early RA signaling restricts the size of the first heart field (FHF) and is required to promote differentiation of second heart field (SHF) at the arterial and venous poles of the developing heart. Here, we sought to elucidate RA-dependent regulatory networks patterning cardiac progenitors within the anterior lateral plate mesoderm (ALPM). A screen for RA-responsive genes within the ALPM of 8 somite (s) stage zebrafish embryos showed that RA loss results in an expansion of six1b and six2a, whose orthologs regulate SHF-derived outflow tract (OFT) development in mice. Quantifying differentiated cardiomyocytes (CMs) in six1b; six2a mutants and treatment with the RA-inhibitor, DEAB, indicated that RA-deficient six1b; six2a double mutants had a reduction in the number of differentiated Nkx2.5⁺ CMs at 48 hours post-fertilization (hpf) compared to RA-deficient six2a single mutant embryos. Previous work has indicated a Tbx1-Six1-Fgf signaling network promotes OFT development in mice. To determine if a similar signaling network functions downstream of early RA signaling, we performed in situ hybridization on Tbx1 knockdown embryos that were treated with DEAB, as tbx1 is also expanded by the 8s stage in RA deficient embryos. We found that the early expansions of six1b and fgf8a were dependent on Tbx1, while the expansion of six2a expression was independent of Tbx1. Together, our data indicate that Tbx1-dependent and independent pathways restrict Six dosage downstream of RA within the ALPM to pattern the CM progenitor field.

Background: Progenitors of cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have not been genetically isolated in the mammalian embryo, making the mammalian SAN one of the last major cardiac structures whose cellular origins remain obscure. We have previously identified an enhancer for Isl1 that is active specifically in PCs during development and maturation of the SAN (Isl1-SAN-Enhancer or ISE). Here, we explored whether ISE could be used as a genetic marker to probe lineage dynamics of early SAN progenitors. 

Methods and Results: ISE-hsp68-CreERT2 transgenic mouse lines were generated with pronuclear injection to facilitate genetic labeling of putative SAN progenitors. Fate mapping with this mouse line showed that ISE-CreERT2⁺ cells contribute to SAN and right atrial myocardium beginning at E8.5, with progressive restriction to SAN fate at later timepoints, raising the possibility that ISE-CreERT2+ cells are initially bipotent at E8.5 and commit to PC or right atrial cardiomyocyte fate between E8.5 and E9.5. To test this possibility, we performed clonal fate mapping using Mosaic Analysis with Dual Markers (MADM), a strategy that relies on mitotic recombination to generate red/green-fluorescent or unlabeled/yellow-fluorescent “twin spots” that can be visually tracked to demonstrate clonal relationships. To do this, we crossed ISE-CreErt2; MADM-11^GT/GT with MADM-11^TG/TG, induced Cre activity with tamoxifen at E8.5, and analyzed clones at E14.5. 30 twin spots have been observed thus far, and immunostaining is underway to accurately determine locations and cellular identities of each twin spot. 

Conclusions:  (1) ISE-CreERT2 mice permit labeling, isolation, and genetic manipulation of PCs and their progenitors. (2) ISE-CreERT2⁺ progenitors at E8.5 contribute to the SAN and portions of the right atrium. (3) Clonal fate mapping will define the potency of these progenitors and the timing of their commitment to PC fate.

During gastrulation, cardiac progenitors (CPs) are identified by the expression of the transcription factor Mesp1. Previous studies have demonstrated that CPs are already spatiotemporally restricted to specific cardiac lineages by that stage. Thus, the heart is built from distinct CPs that display different potential. Recent transcriptional analyses have uncovered the molecular heterogeneity of CPs. In order to understand how CP heterogeneity affects their fate in the adult heart, our aim is to define and modulate the mouse genetic program or “heart code” driving CP specification. We are focusing, in particular, on homeodomain transcription factors that are heterogeneously expressed in CPs. To this end, our objectives are to identify CP sub-populations by defining the spatiotemporal pattern of homeodomain genes during gastrulation and their final cardiac contributions. The role of the homeodomain genes in regulating the pre-specification of CPs are assessed by modulating their expression and analyzing how it impacts CP subpopulations in vivo and in vitro. Our promising preliminary results have already pinpointed the spatial heterogeneity of homeodomain gene expression during gastrulation and have suggested an important role for Lhx1 in Mesp1+ progenitors to correctly form head structures. Together these results will allow the dynamic characterization of heterogeneity within CPs and the identification of putative factors that could trigger the differentiation toward one particular cardiac cell type. These studies have important implications for better understanding the etiology of congenital cardiac malformations and should be the starting point of further applications for improving cell therapy during cardiac repair.

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

The heart of the first land-living vertebrates, tetrapods, is inferred by comparing present-day lungfish and amphibians. These groups belong to lobe-finned fish as do the rare coelacanths. Few coelacanth hearts have been studied and it has been concluded they are very primitive because of a linear chamber topology. Until now, the coelacanth heart has been omitted from inferences of the ancestral tetrapod heart. Using MRI, CT, and 3D-modelling, we assessed the morphology of in-situ hearts of two coelacanths, 13 specimens from all three major groups of amphibians, five specimens from all three major groups of lungfish, nine ray-finned fish, 22 cartilaginous fish, two lampreys, and two hagfish. The coelacanth heart is long mostly due to an elongate outflow tract, but this setting resembles that of fellow ray-finned non-teleost fish. The cardiac chambers have a substantial left-right symmetry as in other bony fish, while the coelacanth sinus venosus and atrium are positioned relatively caudally. Nonetheless, the coelacanth heart is much different from the hearts of cartilaginous fish, lampreys, and hagfish, and it is not primitive. We then assessed 20 gross morphological characters in amphibians, lungfishes, and coelacanths. Nine were shared by all groups. Five were shared by amphibians and lungfishes only, such as a lung vein connecting to the left atrium and an atrial septum. Six were shared by amphibians and coelacanth, such as a valved atrioventricular junction (lungfishes have a ‘plug’) and absent ventricular septum. We conclude the ancestral tetrapod heart likely had a lungfish-like atrium and a coelacanth-like ventricle.

Mechanical properties are cues for many biological processes in health or disease(1). In the heart, changes to the extracellular matrix composition and cross-linking results in stiffening of the cellular microenvironment during development. Moreover, remodelling after myocardial infarction, or in cardiomyopathies lead to fibrosis and a stiffer environment. By combining nanopillar arrays, PDMS gels with defined stiffness and FRET molecular tension sensors, we previously identified a fundamental mechanism for cardiomyocyte rigidity sensing, whereby single cardiomyocyte adhesions sense simultaneous (fast oscillating) cardiac and (slow) non-muscle myosin contractions. Together this results in extracellular matrix dependent levels and dynamics of tension on the mechanosensitive adaptor protein talin(2). To investigate the mechanical signalling further, we analyse force dependent and force-independent talin interactions using specifically designed biofunctionalized nanoarrays, biochemistry and various microscopy approaches(3)(and unpublished data). These experiments surprisingly reveal a mechanical imprinting that is resulting in changes to the talin interactome, cytoskeletal organisation and cardiomyocyte mechanics.

1.         T. Iskratsch, H. Wolfenson, M. P. Sheetz, Appreciating force and shape-the rise of mechanotransduction in cell biology. Nat Rev Mol Cell Biol 15, 825-833 (2014).

2.         P. Pandey et al., Cardiomyocytes Sense Matrix Rigidity through a Combination of Muscle and Non-muscle Myosin Contractions. Dev Cell 44, 326-336 e323 (2018).

3.         W. Hawkes et al., Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control. Faraday Discuss 219, 203-219 (2019).

Introduction: Long QT syndrome (LQTS) is an inherited primary arrhythmia syndrome with a hallmark of QT prolongation on 12-lead ECG. The mechanism of disease is secondary to a delayed repolarization and pathologically prolonged action potential duration of cardiomyocytes. LQTS type 2 (LQT2) is associated with loss-of-function variants in the KCNH2 gene, affecting the rapid rectifier potassium current (IKr). 

Objective: Studying LQTS in an induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model allows for patient-specific phenotype characterization in vitro. We report such a strategy in a LQT2 patient (ECG QTc 492msec) with KCNH2 (c.2464G>A) pathogenic variant, and in a healthy control for comparison (ECG QTc 388msec).

Methods: Human iPSCs were generated from patient and control dermal fibroblasts using a non-integrational Sendai reprogramming method. Beating cardiomyocyte clusters were dissociated and seeded onto Maestro CytoView multi-electrode array plates (Axion BioSystems). Serial corrected field potential duration (FPDc) measurements (n=26), equivalent to QTc values on ECG, were recorded on days 24 to 39 of cardiomyocyte differentiation per sample. Cardiomyocytes were exposed to varying concentrations of Verapamil, Mexiletine, Sotalol, Clarithromycin, and E-4031 through cell culture media exchange. Paired t-tests (non-parametric) were utilized for P<0.05.

Results: Reprogrammed iPSCs displayed pluripotency characteristics. IPSC-CMs had confirmed cardiac lineage. Prolonged FPD values, corrected for beating rate, were observed in the LQT2 patient-derived iPSC-CMs (P<0.0001). Verapamil, Mexiletine, Sotalol, Clarithromycin, and E-4031 altered FPDc in a dose-dependent response.  

Conclusions: Our findings indicated that an iPSC-CM model reliably recapitulates the LQT2 disease phenotype. Drug effects on cardiomyocyte repolarization were demonstratable in the iPSC-CM model.

Cardiopharyngeal mesoderm (CPM) contributes to the formation of the heart and head muscles. While the mechanisms governing CPM specification remains relatively unknown, there is a lack of an in vitro platform that would allow the differentiation of both head and heart muscles. Here, we show that the formation of embryonic organoids from mouse embryonic stem cells (mESC), also called gastruloids, allow the formation of the CPM and its specification toward the cardiac and muscle lineages. By using single-cell RNAseq, and high-resolution imaging of single molecule FISH experiments, we show that the CPM is established in gastruloids, with a kinetic of gene expression that is similar to the mouse embryo. By performing lineage tracing in gastruloids, we further demonstrate that the head and heart muscles formed in this in vitro model derive from CPM progenitors that activates the anterior second heart field specific enhancer of Mef2c. These findings unveil the potential of mESCs-derived gastruloids to undergo CPM specification in both head and heart lineages, allowing the investigation of the mechanisms of CPM specification in development and how this could be affected in congenital diseases.

Cardiac fibroblasts derive from the fetal epicardium and are known to play essential roles in heart development during fetal life and in disease progression in the adult organ.  To model cardiac fibroblast development, we established organoids consisting of hPSC-derived epicardial cells and cardiomyocytes. Within a few days of organoid formation, the two populations segregated in the structures, with the epicardial cells establishing an epithelial layer surrounding the inner population of cardiomyocytes, recapitulating the organization of these two cell types in the fetal heart.   This segregation disappears over the next 10 days of culture as the epicardial cells undergo an epithelial to mesenchymal transition and give rise to fibroblasts and smooth muscle cells that invade the organoid.  To be able to use the organoids to model disease, we treated them with a combination of hormones and activators of fatty acid metabolism to promote the maturation of both the cardiomyocyte and fibroblast populations.  When subjected to pathological stimuli, both the cardiomyocytes and fibroblasts within the mature organoids recapitulated changes observed in the corresponding populations in the failing human heart. Single-cell RNA-seq and RT-qPCR analyses revealed a high degree of fibroblast heterogeneity and identified a unique subpopulation of cells with reparative features in the ‘heart failure’ organoids. Together, these findings demonstrate that it is possible to recapitulate the epicardial-cardiomyocyte interactions in simple organoid structures that lead to the development of cardiac fibroblasts. They also show that these organoids can be used to model aspects of heart failure and enable the identification of specific fibroblast populations that may play specific roles in the progression of heart disease.   

Introduction. The cardiopharyngeal mesoderm (CM) differentiates into endothelial (EC), skeletal and cardiac muscle cells. Our goal is to identify enhancers required for EC differentiation from CM. We have used a serum-free differentiation protocol in mouse ES cells (mESCs) that induces CM differentiation and performed RNA-seq and ATAC-seq to define transcription and chromatin accessibility profiles.
Results. mESCs differentiation induced the expression of EC-specific markers at day 4 of differentiation (d4), including Pecam1, Cdh5, Eng, Kdr, Gata2, Gata6, Ets1, Flt1. RNAseq performed between d2 and d4 identified 1735 differentially expressed genes, many of which are involved in angiogenesis, indicating the activation of an EC transcription program. ATAC-seq revealed 6348 regions that became accessible during this time window. Based on these data, we identified 10 putative enhancers associated with genes involved in EC differentiation. Sequence analyses of regions opened at d4 identified Gata1, Gata2 and JunB transcription factors. We are following two strategies to validate the putative enhancers: DNA editing and epigenetic decommissioning. We first generated mES cells with deletion of putative enhancers of Pecam1 and Notch1, which we then differentiated towards CM-EC lineages. However, Pecam1 and Notch1 expression was unchanged, suggesting that these regions are unrelated to the transcription process. For the second approach, we recently obtained mES cells that stably express dCas9-LSD1 that will be used to test enhancer decommissioning. We performed an EC-specific modification of differentiation protocol, that induced ECs with high efficiency (91%). Pecam1 and Notch1 enhancers will be tested on this new differentiation conditions.
Conclusions. Our ATAC-seq data efficiently identifyied putative endothelial enhancers. Genetic and epigenetic manipulation of these sequences will establish their requirement for EC differentiation from cardiopharyngeal mesoderm.

Cardiac disease is the leading cause of death in the western world and a great proportion of cardiac-related deaths are a result of heart failure or acute myocardial infarction. Good cellular models to mimic primary cardiac cells suitable for drug screening and the generation of disease models are therefore needed. In our lab, we developed a novel protocol for human pluripotent stem cell differentiation into left ventricular-like cardiomyocytes (hPSC-LV-CMs). This protocol results in a homogeneous population of cells with more mature features compared to time matched cells produced by the widely used wnt-on/wnt-off protocol (Standard protocol). Mitochondrial metabolism is an important feature of a mature cardiomyocyte; mitochondria are the main producers of energy in the cardiac tissue, they control calcium handling, signalling and apoptosis. When we sought to understand what happens to mitochondrial function and shape we found that our hPSC-LV-CMs have more mitochondria spread along the myofibers, enhanced respiration and higher mitochondrial membrane potential compared to cardiomyocytes generated using the standard protocol. In addition, hPSC-LV-CMs have more elongated mitochondria, a feature that correlates usually with improved function and increased ATP production. When we grew our LV cardiomyocytes in media rich in fatty acids-the main energy fuel of the adult heart- we were able to further improve mitochondrial spare respiratory capacity, suggesting that modulation of nutrients alone can metabolically mature our LV cardiomyocytes. Fine tuning the maturation media regime will further improve our model and make it more suitable for research aimed at ameliorating LV function and disease phenotypes.

Noonan syndrome (NS) constitutes one of the most common causes of congenital heart defects (CHD). NS infants with mutations in RAF1 present with cardiomyopathy and a variety of CHD. No treatment exists for NS children with CHD or cardiomyopathy, therefore there is an urgent need to understand the mechanisms underlying the disorder to identify new therapies.

Toward that goal, we used hiPSCs as a human cardiogenesis model and made hiPSCs from children with NS carrying mutations in the CR2 domain of RAF1. In addition, we generated isogenic corrected as well as RAF1 KO cells using CRISPR-Cas9dn. To investigate the impact of NS and KO RAF1 on cardiogenesis, we differentiated our isogenic lines towards a cardiac fate. Using flow cytometry for cTNT+ cells at day 15, we first found that NS RAF1 increased cardiac differentiation efficiency (74% vs 58%, n=5, p<0.05), while RAF1 deletion drastically reduced it (15%, n=5, p<0.05), demonstrating that RAF1 regulates cardiac development and NS mutations impair this process. At day 6, cardiac progenitor differentiation measured by flow cytometry for NKX2.5+ cells was diminished in KO cells (14% vs 63%, n=5, p<0.05), while it was potentiated by NS mutations (73%, n=5, p<0.05), suggesting that RAF1 regulates cardiac differentiation early on during development. Using RNA-seq at day 2, we found that NS RAF1 significantly increased expression of mesoderm genes (DLL1, MESP1/2, TBX6), whereas RAF1 KO reduced it. Moreover, GSEA revealed that NS RAF1 was associated with increased expression of EMT (SNAI2, VIM, FBN1) as well as Wnt/β-catenin target genes (TCF7, LEF1, FZD1, DVL2). On the contrary, RAF1 KO led to a strong reduction of the expression of those genes.

Taken together, we show that RAF1 is required for human cardiac differentiation and reveal that NS mutants impair cardiac mesoderm specification by impacting the Wnt/β-catenin pathway. Experiments are in progress to further delineate how RAF1 regulates cardiac mesoderm specification.

Mutations in LMNA, the gene that encodes lamin A/C are the most common cause of familial dilated cardiomyopathy (DCM), often referred as cardiolaminopathy. Despite LMNA being ubiquitously present, the mechanisms that underlie cardiolaminopathy remain elusive. Using induced pluripotent stem cell (iPSCs)-derived endothelial cells (iPSC-ECs), we recently showed that LMNA-induced DCM, due to a frameshift variant caused endothelial dysfunction. Next generation sequencing identified Krüppel-like Factor 2 as the transcription factor responsible for the EC dysfunction, which was reversed by a subset of statins, including lovastatin both in vitro and in vivo. Importantly, iPSC-cardiomyocytes (iPSC-CMs) from LMNA-DCM patients showed improvement in their function when co-cultured with iPSC-ECs and lovastatin, indicating an intricate crosstalk between the ECs and CMs in LMNA cardiomyopathy. Despite impressive progress, little attention has been given to the potential importance of cell-to-cell signaling between ECs and CMs, even though ECs serve a paracrine function to enhance signaling in CMs. To decipher this, we fabricated in vivo-like cardiac 3D engineered heart tissues (EHTs) from LMNA patient’s iPSC-CMs and iPSC-ECs and performed high-resolution assessment of their phenotype. Moreover, single-cell omics on these 3D EHTs revealed an exquisite ligand-receptor (L-R) interaction that we believe to be involved in the EC-CM crosstalk in cardiolaminopathy patients. Indeed, loss of function studies further validated the importance of this L-R pair both in vitro as well as in vivo. Results from this study, which leverages multidisciplinary platforms such as iPSCs, 3D bioengineering models, and single cell-omics, could potentially lead to new therapeutic strategies for DCM patients.

Understanding how the heartbeat starts and is propagated in the human tissue, has been unexplored, because the earliest we can get a recording is well into heart development (around day 26). However, in mice, the developing heart starts beating earlier during the formation of the cardiac crescent (similar to day 16 in humans). During this early stage of development, the propagation of signal undergoes a complex shift, in the initiation of the beat, before the pacemakers have formed. Here we use human self-organizing cardioids to decipher the ontogeny of early heartbeats by tracking contraction, field potentials, and calcium transients for different compartments of the heart, including the left ventricle, right ventricle, atria, and atrioventricular canal.  We find that each compartment has distinct characteristics which develop differently over time. With this knowledge we will be able to gain deeper understanding of the mechanisms of the initiation and propagation of human heartbeats.

During development, the epicardium contributes to the heart’s cellular components and guides and supports expansion of the vascular network. A multitude of coordinated heparan sulfate proteoglycan (HSPG)-dependent pathways control epicardial cell activity. Sulf1 and Sulf2 - enzymes that modify HSPGs – present another level of control to refine signalling separate to ligand presence. WT1 is required for heart development and denotes the epicardium’s activation state both in the embryonic and injured heart. Wt1 and Sulf1 are enriched in the activated adult epicardium post-injury. Regulation of Sulfs and their specific roles in the epicardium are unknown. Here, we used in situ hybridisation techniques and scRNA-seq to investigate Sulfs over the course of embryonic mouse heart development. In addition, we carried out ATAC-seq to investigate open chromatin in the Sulf genes, and to identify potential WT1 transcription factor occupancy. Our study shows Sulf1 is expressed in epicardial progenitors and the forming epicardium, whilst Sulf2 is expressed in cardiomyocytes during early cardiac development. Sulf1 strongly co-localises with Wt1 expression, with levels further coinciding with loss of Wt1 as the epicardium quiesces or undergoes epiEMT. Wt1 null embryonic hearts, which form an irregular epicardial layer, demonstrated lower levels of Sulf1 and upregulated Sulf2. We found that WT1 binds to the Sulf1 promoter, but no binding was detected within the Sulf2 gene in E13.5 epicardial cells. Thus, while WT1 directly regulates Sulf1 expression, upregulation of Sulf2 instead occurs indirectly. Our findings show WT1 differentially regulates Sulfs to mediate HSPG-dependent signalling in the embryonic epicardium.

microRNAs (miRNAs) are an evolutionary conserved regulatory RNAs involved in many biological processes, including heart development as well as cardiac disease. miRNAs of the let-7 family are highly prevalent in the heart, and their expression is altered in several cardiovascular diseases, such as dilated cardiomyopathy and arrhythmia. Overexpression of let-7 was promotes hPSC-derived cardiomyocyte maturation and adult-like metabolism. In humans, the let-7 family consists of 12 paralogous miRNAs with largely redundant function, which has been limiting the identification of target genes and understanding let-7’s role in establishing or maintaining heart function.

To explore the role of let-7 we used the Drosophila model that only has a single let-7 miRNA family member. We found that in flies let-7 expression is enriched in the heart, and loss of let-7 causes cardiac dilation, systolic dysfunction, and arrhythmia, indicating that let-7 is a critical for many aspects of heart function. To identify let-7 effector genes we performed proteome analysis on flies where let-7 was depleted in cardiac and somatic muscle. We found a very specific response of gene products involved in lipid and glucose metabolism, such as APOB/Apolpp and SORD/Sodh-1, as well as signaling proteins like TGFB1/Dawdle and BMPR1B/Thickveins. Most responding proteins seemed to be highly enriched in the fat body, the fly’s adipose tissue, which points to non-autonomous consequences of loss of let-7 in heart and muscle on the adipose tissue.

We also cross-validated these findings with single-cell sequencing data from female and male adult fly tissues and were able to confirm that most proteins identified in our proteomics study are indeed transcribed in the fat body, many of them being differentially expressed between female and male Drosophila tissues. We therefore hypothesize that let-7 might also serve as a switch that controls tissue metabolism downstream of sex-specifying pathways

Congenital heart disease is an independent risk factor for arrhythmias and cardiac remodelling which leads to heart dysfunction and failure. Heart failure remains one of the main causes of death worldwide. It is associated with cardiomyocyte death, a mechanism that is not fully understood. The zinc-finger transcription factor GATA4, a known regulator in cardiac development, is essential to circumvent cell death along with the AMPK/Sirtuins/PGC1α energy sensor pathway. In collaboration with a medicinal chemistry lab, LCB-2122 was synthesized to target survival pathways. LCB-2122 enhanced ejection fraction in mice overexpressing angiotensin receptor type 1 (AT1R) and mice treated with the chemotherapeutic agent Doxorubicin (DOX). In AT1R mice, LCB-2122 improved mitochondrial structure and rescued against DOX-induced cell death. Here, I aim to decipher the mechanism of action of LCB-2122. Interestingly, ejection fraction was also enhanced in GATA4 heterozygous mice treated with DOX. We hypothesize that LCB-2122 enhances mitochondrial function and activates energy-producing pathways. In primary cardiomyocytes, LCB2122 abolished the cell death caused by DOX and increased phosphorylation of AMPK and its downstream target acetyl Co-A carboxylase. This effect is proven to be AMPK dependent. In-vitro, the presence of LCB-2122 augmented Sirtuins 1 and 3 activities. MitoSOX-Red shows that LCB-2122 decreases DOX-induced mitochondrial superoxide. Also, LCB-2122 increases GATA4 levels and rescues the DOX-induced GATA4 depletion. We will next use unbiased approaches such as the metabolomics and photoaffinity experiment to guide us to new direct binding partners to LCB-2122. Furthermore, other LCB-2122 derivatives are synthesized and tested to study the structure-function relationship to enhance bioactivity and stability. This project will introduce a new potential drug that protects against cardiotoxicity by activating cardiomyocyte survival pathways.

Endothelial damage is directly associated with various cardiovascular diseases, including stroke and ischemic heart disease, the two leading causes of death globally.  Previous studies showed that vascular regeneration involves a subset of regenerating endothelial cells (ECs) with distinct proliferative ability and molecular signature.  However, the specific molecular mechanisms driving vascular regeneration remain elusive.   

Here, we establish and characterize a zebrafish vascular regeneration model by utilizing nitroreductase-mediated EC ablation at embryonic and larval stages.  By following the regenerating ECs during regeneration using state-of-the-art high-resolution time-lapse imaging, we observed that certain ECs are more proliferative, sprout better, and reconnect with the neighboring ECs.  These data suggest a heterogeneous response to tissue damage by some EC subpopulations, which possess a higher capacity of repopulating the vasculature.  To delineate this heterogeneity, we performed single-cell RNA-sequencing after EC ablation and recovered 6051 cells with 8 major cell types from the larval caudal region.  We further analyzed 1929 ECs and found two sub-clusters displaying a higher proliferative capacity.  One of these proliferative EC sub-clusters expresses genes involved in Notch signaling, leading us to hypothesize that it is involved in vascular repair.  We now aim to unravel the molecular and cellular features of these proliferative EC sub-clusters by performing cell-specific manipulations in vivo.  Overall, our findings can help us gain a better understanding of the gene programs critical for vascular repair and regeneration, which may have therapeutic implications.

After myocardial infarction (MI), the myocardium heals through a process of scarring, in which previously healthy muscle is replaced by fibrous tissue. Remarkably, fish and neonatal mice do not heal by permanent scarring but are able to fully regenerate the heart. Immune cells are essential to this process, though their precise roles are complex and not fully understood.

In order to resolve this complexity we performed scRNA-seq analysis of the murine neonatal heart before and after injury. This analysis allowed us to identify specific genes and pathways involved in the different reparative processes acting in neonatal pro-regenerative (P1) and pro-fibrotic (P7) hearts. Interestingly, differential gene expression and gene set enrichment analysis indicated changes in the P1 granulocytes population such as highly significant enrichment in cysteine endopeptidase inhibitory activity (Stefin A1), dampened inflammation and enriched extracellular matrix (ECM) remodeling. We hypothesized that the inhibition of catalytic enzymes involved in matrix  remodeling in P1 hearts might play a role in altering the structure of the scar tissue. To test the hypothesis, we used a commercially available cysteine endopeptidase inhibitor (E64) to inject P7 mice after MI. The in vivo experiment showed structural changes in the scar formation at 21 days post-injury with a significative reduction in the coefficient of alignment of collagen fibers. These structural changes might lead to the formation of a less compact, more unstable scar which could be more easily resorbed thus creating a more permissive regenerative environment.

In conclusion, here we identify new fibrotic pathways that may be targeted to extend the regenerative capacity of the heart beyond neonatal stages into adult life. 

Tbx5 and Mef2c encode critical cardiac transcription factors that are necessary for proper development of the looped heart tube. In Tbx5 knockout (KO) mice, the sinoatrial structures and primitive left ventricle are severely hypoplastic at E9.0. Conversely, Mef2c KO leads to the apparent absence of the right ventricle and hypoplastic outflow tract at E9.0. These two complementary, mutant phenotypes provide a suitable platform with which to dissect the genomic regulation of heart tube morphogenesis. To gain a more complete understanding of the genomic regulators underlying heart tube formation, we have performed combined single-nucleus RNA- and ATAC-sequencing on wild type, Tbx5 KO, and Mef2c KO embryos at E7.75 (cardiac crescent) and E9.0 (looped heart tube) using the 10x Genomics Multiome system. Initial analysis of gene expression data revealed a delay in cardiomyocyte differentiation in Mef2c KO mice at E7.75. Additionally, we found an expansion of atrial and atrioventricular cushion markers in the Mef2c KO cardiomyocytes that formed by E9.0. We are currently integrating the ATAC-seq data to determine changes in chromatin accessibility that underlie the improper specification of cardiac progenitors. Next, we will conduct live embryo imaging of wild type and mutant embryos expressing fluorescent markers that demarcate first and second heart field lineages to discern precisely when and where linear heart tube formation goes awry. Together, these studies will provide fundamental insights into how the mammalian heart takes shape during embryogenesis.

Activated cardiac fibroblasts (CFs) are responsible for the healing of the heart tissue after a myocardial infarction (MI). Based on high throughput technologies, several groups have recently demonstrated their heterogeneity and a unique role of each subpopulation of CFs during the ventricular remodelling process. This is relevant towards the discovery of personalized treatments to control the initial post-MI healing scar that will contribute to preserve ventricular function and prevent the onset of heart failure. However, little is known about the moment that CFs are activated, and which genes are potentially involved in this process. Using a mouse model for MI and single cell RNA-Seq, we demonstrate that the activation of Reparative Cardiac Fibroblasts (RCFs), the CFs responsible for the healing scar, happens within the first week after MI. Interestingly, our data reveals that all CFs show high expression of the top markers genes for RCF in a specific moment, but only few of them finally evolve to an RCF transcriptomic identity. Furthermore, we describe two different molecular dynamics that could give rise to this activation and, in consequence, the appearance of definitive RCFs. Using Spatial Transcriptomics, we localized the genes related to each dynamic in different anatomical regions of the infarcted heart, but, remarkably, only one persists seven days after MI. These results highlight the existence of a specific “window of activation” of RCFs at the beginning of the ventricular remodelling process. This potential ´therapeutical window´ could allow us to regulate the size of the healing scar and, in consequence, the poor prognosis for patients that have suffered an ischemic event. 

Reconstructing the circuits that control how cells adopt specific fates and engineering these circuits to reprogram cellular functions are major challenges in biology. I will introduce a series of experimental and computational frameworks such as “Waddington-OT”, “Raman2RNA” for reconstructing molecular dynamics over time and in live cells through single-cell multi-omics and imaging. I will introduce how we can use these approaches to decode the cellular and molecular mechanisms governing reprogramming and development. 

In the vertebrate heart, the leading pacemaker role is held by the sinoatrial node (SAN), which is at the origin of each heartbeat. This crucial function originates in the capacity of the pacemaker cells to spontaneously generate action potentials that propagate in an orderly manner across the surrounding cardiac tissue and eventually trigger the synchronized contraction of the heart chambers. The SAN not only displays great variability in size, shape and architecture across vertebrate species, but its complexity increases as development progresses, going from a bundle of spontaneously depolarizing cardiomyocytes to a heterogenous tissue comprising amongst others fibroblasts and cells of neural origin, additionally to specialized cardiomyocytes. How this structure comes together remains unclear, yet its conservation suggests that its assembly is driven by a set of general, elementary principles.

We have carried out single-cell RNA sequencing on cardiomyocytes of zebrafish at two days post-fertilization, the developmental stage at which pacemaker function is first observed. Having identified the cells harboring pacemaker activity, we are currently examining their transcriptomic signature to get more insight into the molecular processes leading to their establishment. Additionally, through analysis of genes differentially expressed in this specific cardiomyocyte sub-population, we hope to get better understanding of the integration of the pacemaker in cardiac function.        

A primary limitation in the clinical application of pluripotent stem cell derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, perinatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Here, we used our previously developed large particle fluorescence-activated cell sorting approach to generate an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. We subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through trajectory reconstruction methods, we identified a perinatal maturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of our trajectories with previously published human datasets, we identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, we demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level, and can be leveraged towards improving the clinical viability of PSC-CMs.

The CDDRC provides an innovative cloud-based platform to facilitate the analysis, visualization and sharing of genomic data from research in cardiac development and regeneration in several species. Leveraging institutional investments in genomics and personalized medicine, the CDDRC will facilitate dynamic and investigator-interactive cloud-based interfaces with multiple -omics datasets from humans and multiple model organisms, resulting in collaborative discoveries of the genetic and epigenetic causes of congenital heart disease (CHD). In addition, our program will have educational components in Ethical, Legal and Social Implications (ELSI) of CHD genetics and genomics, and an outreach program in CHD computational biology for students from underrepresented groups. The CDDRC will bring together researchers with a diverse range of expertise, with the goal of understanding the causes of congenital heart disease in children.

The CDDRC is the newest component of the NHLBI Bench-to-Bassinet (B2B) program (https://benchtobassinet.com), along with the Pediatric Cardiac Genomics Consortium (PCGC) and the Pediatric Heart Network (PHN). The goals are 1) to populate the platform with multi-omics data (single cell-omics, RNA-seq, ATAC-seq, ChIP-seq, Hi-C, etc.) from multiple organisms from the previous basic science component of the B2B, the Cardiovascular Development Consortium (CvDC), 2) to recruit new datasets from the cardiovascular community to the platform, and 3) to develop, recruit and incorporate new bioinformatics analytic and visualization tools to the platform, and 4) intersect this cloud-based platform with human CHD genetics datasets and the NHLBI BioData Catalyst (BDC) initiative.

At the Weinstein meeting we will be announcing Challenge Prize competitions that will fund new projects in the CDDRC from outside investigators, and a new CDDRC Fellowship program that will provide training opportunities for outside investigators in cloud-based genomics computation.

Left ventricular non-compaction (LVNC) associated with dilation is characterized by the coexistence of left ventricular dilation and systolic dysfunction. Pediatric cases with dilated-LVNC have worse outcomes than those with isolated dilated cardiomyopathy and adult patients. Herein, we report a pediatric case with early-onset of dilated-LVNC.

Trio-based whole-exome sequencing revealed compound heterozygous variants in SPEG gene, involving one common de novo missense variant and one ultra-rare paternally inherited variant. Both patient's sisters, as well as his father who presented LVNC without cardiac dysfunction harbor the ultra-rare SPEG variant only.

The current study adds further evidence on the cumulative effect of SPEG variants as a mechanism of LVNC cardiomyopathy, the pleiotropic nature of this gene, and the clinical relevance of de novo mutations.

Bicuspid aortic valve (BAV) is the most common congenital heart defect, affecting up 0.5% to 2% of the population. BAV is characterized by the presence of two leaflets instead of three symmetrical ones in normal aortic valves. BAV patients usually develop premature CAVD leading to valve stenosis that requires valve replacement. Recent studies have shown that Gata6 haploinsufficient mice show RL-type BAV phenotype, the most common in humans. Proliferation, differentiation and migration processes are affected in Gata6^+/- mice giving rise to OFT defects which lead to BAV malformation. We previously showed that abrogation of the Notch regulator Mib1 causes BAV.  Gata6 and Mib1 are located in chromosome 18, and only 300Kb apart. We have generated three different mouse lines using CRISPR-Cas9, combining Gata6 nonsense mutation with different Mib1 nonsense and missense mutations to examine the possibility of a common regulation of Gata6 and Mib1 in valve morphogenesis.  Double heterozygous mice with Gata6 and Mib1 in trans configuration show reduction in BAV penetrance to 40%, compared to 70% in Gata6^+/- mice. Additionally, double heterozygous mice with Gata6 and Mib1 in cis configuration show a complete suppression of the BAV phenotype. In contrast, combination of Gata6 with Notch1 mutations does not increase or reduce the 70% penetrance of BAV in double heterozygous mice, indicating that the observed BAV reduction is Mib1-dependent. Gata6 and Mib1 belong to the same topologically associating domain (TAD) and we will try to study the regulatory implications that this has and its impact in valve morphogenesis.

Bicuspid aortic valve (BAV) is the most common congenital heart defect with a high index of heritability. Patients with BAV have different clinical course and disease progression.

Herein, we report three siblings with clinically heterogeneous BAV. Their clinical presentations include moderate to severe aortic regurgitation, aortic stenosis, and ascending aortic aneurysm. Genetic investigation was carried out using Whole Exome Sequencing for the three patients.

We identified two non-synonymous variants in ROBO1 and GATA5 genes. The ROBO1: p.(Ser327Pro) variant is shared by the three BAV affected siblings. The GATA5: p.(Gln3Arg) variant is shared only by the two brothers who presented BAV and ascending aortic aneurysm. Their sister who is affected by BAV without aneurysm does not harbor the GATA5: p.(Gln3Arg) variant. Both variants were absent in the patients’ fourth brother who is clinically healthy with tricuspid aortic valve.

To our knowledge, this is the first association of ROBO1 and GATA5 variants in a familial BAV with a potential genotype/phenotype correlation. Our findings are suggestive of the implication of ROBO1 gene in BAV and GATA5: p.(Gln3Arg) variant in ascending aortic aneurysm.

Our family-based study further confirms the intrafamilial incomplete penetrance of BAV as well as the complex pattern of inheritance of the disease.

Cardiovascular morphogenesis involves dynamic behaviors of endocardial cells, which respond to blood flow by orchestrating cell orientation, proliferation, migration and cell shapes. How endocardial cells of the zebrafish heart influence blood flow-responsive behaviors such as cell orientation during cardiac chamber development or ingression into the cardiac jelly during valvulogenesis remains unclear. Here, we show that blood flow induces the expression of the transmembrane ligand Efnb2a specifically within the ventricular endocardium of the nascent heart tube and that its activity is necessary and sufficient to reorient these endocardial cells against blood flow. During valvulogenesis, Efnb2a instructs the reorientation of atrioventricular cushion endocardial cells out of the plane of the endocardium prior to their ingression into the cardiac jelly. In the absence of blood flow, forced and clonal overexpression of Efnb2a within endocardial cells is sufficient to trigger their reorientation, thus establishing Efnb2a as a cell-autonomous morphogenetic regulator that mediates endocardial cell orientation in response to blood flow. 

Developmental etiologies causing complex congenital aortic root abnormalities are unknown. Here we show that deletion of Sox17 in aortic root endothelium in mice causes underdeveloped aortic root leading to a bicuspid aortic valve due to the absence of non-coronary leaflet, as well as mispositioned left coronary ostium. The respective defects are associated with reduced proliferation of non-coronary leaflet mesenchyme and aortic root smooth muscle derived from the second heart field progenitor cells. Mechanistically, SOX17 occupies a Pdgfb transcriptional enhancer to promote its transcription, whereas Sox17 deletion inhibits the endothelial Pdgfb transcription and PDGFB growth signaling to the non-coronary leaflet mesenchyme. Restoration of PDGFB in aortic root endothelium rescues the non-coronary leaflet and left coronary ostium defects in Sox17 nulls. Altogether, these observations support a SOX17-PDGFB axis underlying aortic root development that is critical for aortic valve and coronary ostium patterning, thereby informing a potential shared disease mechanism for concurrent anomalous aortic valve and coronary arteries.  

The global burden created by congenital heart defects (CHD) is firmly established. Whilst the cause of approximately 80% of CHD is unknown, it is thought likely that there is a tightly regulated genetic network that exists to shape development in the great vessels of the heart. These are derived from the pharyngeal arch arteries (PAAs) which form sequentially in mid-embryogenesis, before remodelling to form the mature vessels. One gene implicated in this network, Paired-box gene 9 (Pax9), plays a key role in developmental processes through functional interaction with Tbx1 in the pharyngeal endoderm for 4th PAA development. The extracellular matrix (ECM) exists as a 3D domain that provides structural and biochemical support to surrounding cells and has been found to facilitate the migration of neural crest cells (NCCs) into the pharyngeal arches and influence their subsequent differentiation into smooth muscle cells to support the developing PAAs. Previous studies have demonstrated that cardiovascular defects are present in mice mutated for ECM genes. RNA-seq data has identified ECM organisation as a significantly differentially expressed signalling pathway between Pax9+/+ and Pax9-/- embryos, highlighting the genes involved as candidates for further analysis. Furthermore, immunostaining of key ECM proteins has shown variable expression between Pax9 control and mutant embryos. Studies are underway to validate the expression of these key ECM genes and uncover the mechanisms by which they interact with Pax9 to increase the understanding of how these genetic interactions influence migratory NCC behaviour and the overall effect this may have on cardiovascular morphogenesis. 

Cardiac valves ensure unidirectional blood flow through the heart, and altering their function can result in heart failure.  Flow sensing via wall shear stress and wall stretching through the action of mechanosensors can modulate cardiac valve formation.  However, the identity and precise role of the key mechanosensors and their effectors remains mostly unknown.  Here, we genetically dissect the role of Pkd1a and other mechanosensors in atrioventricular (AV) valve formation in zebrafish and identify a role for several pkd and piezo gene family members in this process.  We show that Pkd1a, together with Pkd2, Pkd1l1, and Piezo2a, promotes AV valve elongation and cardiac morphogenesis.  Mechanistically, Pkd1a, Pkd2, and Pkd1l1 all repress the expression of klf2a, klf2b, and egr1 transcription factor genes implicated in AV valve development.  Furthermore, we find that the calcium-dependent protein kinase Camk2g is required downstream of Pkd function to repress klf2a expression.  Altogether, these data identify and dissect the role of new mechanosensors required for AV valve formation, thereby broadening our understanding of cardiac valvulogenesis.

Heart valve defects are present in ~1/3 of congenital heart disease cases, the most common form of human birth defects. Many affected individuals will require valve replacement. Generation of various valve cell populations in vitro from human pluripotent stem cells (hPSC) would provide unique opportunities to study human valve disease as well as  engineer biological valves. During embryonic development the cell populations comprising heart valves derive from endocardium, a specialized endothelial cell population that lines the chambers of the heart and  induces the formation of the first functional population of cardiomyocytes, known as trabecular cardiomyocytes. To access human endocardial cells, we developed a protocol that promotes the generation of NKX2-5⁺CD31⁺ endothelial cells from hPSC-derived cardiovascular progenitors by manipulating BMP10 signaling. These cells express the cohort of genes that identifies the endocardial lineage in vivo and show a high level of transcriptional similarity to primary human fetal endocardium. Furthermore, these hPSC-derived  NKX2-5⁺CD31⁺ endocardial-like cells display the ability to induce a trabecular fate following co-culture with target cardiomyocytes, and the capacity to undergo EndoMT to give rise to mesenchymal cells that express markers of valvular interstitial cells (VICs). Further culturing of these VIC-like cells resulted in their segregation into two subpopulations secreting different amounts of collagens and proteoglycans/glycosaminoglycans, recapitulating the heterogeneity observed in the stratified extracellular matrix of normal valves. In summary, the findings presented in this report describe a method for the derivation of valvular interstitial cells from hPSCs.

The organization of extracellular matrix (ECM) components, including collagens, proteoglycans, and elastins, into the fibrosa, spongiosa, and ventricularis layers respectively, is essential for maintaining structure and function of heart valves. Mutations in ECM components cause connective tissue disorders, including osteogenesis imperfecta (OI), and debilitating heart valve dysfunction in these patients is common. Currently, treatment is limited to surgical valve repair or replacement, causing insuperable complications for many patients, emphasizing a critical need for developing alternative approaches. This study models connective tissue disorders in mice to investigate valve pathogenesis, with the goal of developing more effective, mechanistic-based therapies.

Mice with a frameshift mutation in the pro-alpha2 chain of collagenI (col1a2) serve as a model of OI Murine (OIM). By histology, aortic valve (AoV) cusps from OIM^-/- mice show significant thickening and myxomatous features, including increased proteoglycan deposition and disorganization of the ECM, beginning at 3 months of age that worsen over time. Ultrastructural electron microscopy of AoV cusps reveal a trend towards decreased area and diameter of collagen fiber cross sections in the OIM^-/- by 9 months. However, we observe no functional defects under echocardiography until 12-14 months, when 67% of OIM^-/- mice exhibit AoV regurgitation and/or left ventricle enlargement, or AoV stenosis. Mass spectrometry of decellularized AoV isolates collected at 3 months of age confirmed decreased ColIa2 in OIM^-/- mice, and identified significant changes in the overall ECM niche as a result of the col1a2 mutation that likely contribute to the observed valvular phenotypes.

Together these studies provide mechanistic insights into the temporal pathogenesis of valve dysfunction associated with human connective tissue disorders.

During cardiac morphogenesis, blood exits the ventricle through the outflow tract and pharyngeal arch arteries (PAAs). Hemodynamics plays a vital role in the maturation of PAAs into the great vessels of the heart. Disruption of established flow patterns during critical windows of development produces a range of cardiac malformations that drastically alter function of the mature heart. Malformation of the great vessels often occur in tandem with outflow tract valve malformations. However, the timing of which defect precedes the other and the role of hemodynamics in disease propagation remains poorly understood. Here, we alter intracardiac flow patterns through early valve disruption. With the chick embryo annimal model we preform highly targeted non-invasive microsurgeries on prevalvular cushions and detail the effects on subsequent PAA morphogenesis. Through the use of two-photon microscopy guided femtosecond laser pulses, we nucleate and control the growth of microbubbles within the developing outflow tract, focally displacing early valve tissue until a permanent ablation that prevents closing of the valve is obtained. We monitor hemodynamic changes through Doppler ultrasound and detail subsequent structural changes using nano-computed tomography. Proximal cushion ablations performed at HH18 (day 3) show instantaneous flow changes that lead to structural changes within 24 hours. We follow these morphological changes in 24-hour increments through HH31 (day 7), detailing arch specific changes in length, curvature and cross-sectional area as well as abnormal aortic sac connections. With these tools, we directly link focal obstructions of the outflow tract to abnormal PAA remodeling that results from local tissue and hemodynamic malformations.

Quantitative PCR (qPCR) aims to measure DNA or RNA concentrations in diagnostic and biological samples based on the quantification cycle (C_q) value observed in the amplification curves. Results of qPCR experiments are mostly reported as Cq, ΔCq, or ΔΔCq values and thus assume that all assays are 100% efficient.

The large variation in reported C_q values among laboratories can be explained readily by differences in qPCR machines, amplification protocols and the setting of the quantification threshold, at the start or at the end of the exponential phase of the amplification curve.

Qualitatively, a reaction that shows specific amplification should be deemed positive, regardless of the observed C_q. Quantitatively, it should be taken into account that the observed C_q is highly dependent on the amplification efficiency. Because this efficiency can vary among targets and samples, meaningful diagnostics or biological interpretation of target quantity and relative gene expression values require that the actual efficiency of the PCR is used in the calculations.

PCR efficiency is frequently derived from standard curves, but this approach is affected by serial dilution errors and hampered by properties of the standard and the diluent. This affects accurate quantification of clinical and biological samples in diagnostic applications and collected in challenging conditions. Determining the mean PCR efficiency from the individual amplification curves per assay avoids these biases in relative quantification.

To take the analysis to the level of absolute quantification and obtain unbiased efficiency-corrected results, we recommend quantification with a single calibrator with known target concentration and efficiency values determined from the amplification curves of the reactions of this calibrator and the unknown samples.

Using this efficiency-corrected approach will help to remove inter-laboratory variability in reported expression levels of genes within the same biological context.