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The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.

Gene deletion by the Cre-Loxp system has facilitated functional studies of many critical genes in mice, offering important insights and allowing deeper understanding on the mechanisms underlying organ development and diseases, such as heart development and diseases. In this study, we generated a Myh6-Cre knockin mouse model by inserting the IRES-Cre-wpre-polyA cassette between the translational stop codon and the 3′ untranslated region of the endogenous Myh6 gene. By crossing knockin mice with the Rosa26 reporter lines, we found that Myh6-Cre targeted cardiomyocytes at the embryonic and postnatal stages. In addition, we were able to inactivate the desmosome gene Desmoplakin (Dsp) by breeding Myh6-Cre mice with a conditional Dspflox knockout mouse line, which resulted in embryonic lethality during the mid-term pregnancy. These results suggest that the new Myh6-Cre mouse line can serve as a robust tool to dissect the distinct roles of genes involved in heart development and function.

Lack of conal rotation and conal malseptation is a characteristic anatomical feature for TOF which lead to dextroposed position of aorta and significant RVOT narrowing. The quantitative assessment of these anatomical features using modern cardiac imaging modality has been rarely discussed in the literature. All TOF scanned had in our center from 2013 till 2019 were included. The angle of aortic root rotation was recorded by measuring the angle between a line connecting the midpoint of the non-coronary sinus to the anterior commissure and another line along the interatrial septum. Rotation angles were correlated with proximal main pulmonary artery (MPA) size indexed to BSA. 287 TOF patients were included, 258 patients (91%) had TOF with pulmonary stenosis (TOF-PS) including 138 male (54%), median age 2 years (2 months–40 years), and 29 patients (9%) had TOF with pulmonary atresia (TOF-PA) including 17 male (59%), median age 5 years (1 m-33 years). The whole cases demonstrated clockwise rotation of the aortic root. The mean rotation angle in TOF-PS group was 52.6 ± 20.9° and in TOF-PA group was 64.9 ± 13.9°. Proximal MPA diameter was 11.1 ± 5.9 mm/m2. There was a significant negative correlation between aortic root rotation angle and proximal MPA diameter (r = − 0.262, P = 0.000). The rotation angle of aortic root was significantly higher in TOF-PA compared to TOF-PS (64.9 ± 13.9° vs. 52.6 ± 20.9°, P = 0.001, respectively). MSCT provide a quantitative measurement methodology of conal malseptation and its effect in TOF patients. There is a clockwise rotation angle of the aortic root in TOF patients that correlates negatively with proximal MPA size. TOF-PA have a larger rotation angle of aortic root.

Force enhancement is one kind of myogenic spontaneous fasciculation in lengthening preload striated muscles. In cardiac muscle, the role of this biomechanical event is not well established. The physiological passive property is an essential part for maintaining normal diastole in the heart. In excessive preload heart, force enhancement relative erratic passive properties may cause muscle decompensating, implicate in the development of diastolic dysfunction. In this study, the force enhancement occurrence in mouse cardiac papillary muscle was evaluated by a microstepping stretch method. The intracellular Ca2+ redistribution during occurrence of force enhancement was monitored in real-time by a Flou-3 (2 mM) indicator. The force enhancement amplitude, the enhancement of the prolongation time, and the tension–time integral were analyzed by myography. The results indicated that the force enhancement occurred immediately after active stretching and was rapidly enhanced during sustained static stretch. The presence of the force and the increase in the amplitude synchronized with the acquisition and immediate transfer of Ca2+ to adjacent fibres. In highly preloaded fibres, the enhancement exceeded the maximum passive tension (from 4.49 ± 0.43 N/mm2 to 6.20 ± 0.51 N/mm2). The occurrence of force enhancement were unstable in each static stretch. The increased enhancement amplitude combined with the reduced prolongation time to induce a reduction in the tension–time integral. We concluded that intracellular Ca2+-synchronized force enhancement is one kind of interruption event in excessive preload cardiac muscle. During the cardiac muscle in its passive relaxation period, the occurrence of this interruption affected the rhythmic stability of the cardiac relaxation cycle.

Total anomalous pulmonary venous connection (TAPVC) is a rare congenital cardiac malformation, and prenatal detection of TAPVC malformation remains a challenging. TAPVC can be easily missed or misdiagnosed in prenatal examinations. This study was aimed to use the modified vascular corrosion casting technique to prepare fetal cardiovascular casts with TAPVC and investigate the utility of cardiovascular casting for the demonstration of fetal TAPVC. The retrospective study enrolled twenty fetuses (22 to 29 + 4 gestational weeks) with TAPVC diagnosed by prenatal echocardiography and casting technique from May 2015 to May 2020. Pre- and postnatal medical records, including results obtained by prenatal ultrasound, postpartum computed tomography angiography, as well as anatomic and cardiovascular casting findings were carefully reviewed and analyzed. In twenty cases, 80% (16/20) had intra- or extracardiac malformations. The TAPVC types were supracardiac (n = 8), cardiac (n = 6), infracardiac (n = 4), and mixed (n = 2). The diagnosis of 1 case each of supracardiac and cardiac TAPVC was modified to partial anomalous pulmonary venous connection; additionally, 4 malformations were missed and 2 were misdiagnosed, including an anomalous left brachiocephalic vein in supracardiac TAPVC, abnormal inflow of the hepatic vein and a double inferior vena cava in infracardiac TAPVC; and bilateral ductus arteriosus in infracardiac TAPVC; a tetralogy of Fallot in cardiac TAPVC that was corrected to right ventricular double outlet; and an absence of ductus arteriosus that was misdiagnosed as slim ductus arteriosus. Comparing with ultrasound, casting technique has its own superiority in exhibiting TAPVC abnormalities, especially in certain types such as course, origin and absence abnormalities of ductus. Postpartum cardiovascular casts can accurately depict the branch structure of the heart’s larger vessels, and may be used as a clinical assessment and teaching method in complex cardiac malformations.

Efficient differentiation of pluripotent stem cells (PSCs) into cardiac cells is essential for the development of new therapeutic modalities to repair damaged heart tissue. We identified a novel cell surface marker, the G protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), specific to cardiac progenitor cells (CPCs) and determined its functional significance and therapeutic potential. During in vitro differentiation of mouse and human PSCs toward cardiac lineage, LPAR4 expression peaked after 3-7 days of differentiation in cardiac progenitors and then declined. In vivo, LPAR4 was specifically expressed in the early stage of embryonal heart development, and as development progressed, LPAR4 expression decreased and was non-specifically distributed. We identified the effective agonist octadecenyl phosphate and a p38 MAPK blocker as the downstream signal blocker. Sequential stimulation and inhibition of LPAR4 using these agents enhanced the in vitro efficiency of cardiac differentiation from mouse and human PSCs. Importantly, in vivo, this sequential stimulation and inhibition of LPAR4 reduced the infarct size and rescued heart dysfunction in mice. In conclusion, LPAR4 is a novel CPC marker transiently expressed only in heart during embryo development. Modulation of LPAR4-positive cells may be a promising strategy for repairing myocardium after myocardial infarction.

During cardiogenesis, the outflow tract undergoes a complicated morphogenesis, including the re-alignment of the great blood vessels, and the separation of aorta and pulmonary trunk. The deficiency of FGF8 in the morphogenesis of outflow tract has been well studied, however, the effect of over-dosed FGF8 on the development of outflow tract remains unknown. In this study, Rosa26R-Fgf8 knock-in allele was constitutively activated by Wnt1-cre transgene in the mouse neural crest cells presumptive for the endocardial cushion of outflow tract. Surprisingly, Wnt1-cre; Rosa26R-Fgf8 mouse embryos exhibited persistent truncus arteriosus and died prior to E15.5. The cardiac neural crest cells in Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus did not degenerate as in WT controls, but proliferated into a thickened endocardial cushion and then, blocked the blood outflow from cardiac chambers into the lungs, which resulted in the embryonic lethality. Although the spiral aorticopulmonary septum failed to form, the differentiaion of the endothelium and smooth muscle in the Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus were impacted little. However, lineage tracing assay showed that the neural crest derived cells aggregated in the cushion layer, but failed to differentiate into the endothelium of Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus. Further investigation displayed the reduced p-Akt and p-Erk immunostaining, and the decreased Bmp2 and Bmp4 transcription in the endothelium of Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus. Our findings suggested that Fgf8 over-expression in cardiac neural crest impaired the formation of aorticopulmonary septum by suppressing the endothelial differentiation and stimulating the proliferation of endocardial cushion cells, which implicated a novel etiology of persistent truncus arteriosus.

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice., Heart-forming organoids model early cardiac development.

Bmp plays an important role in cardiomyocyte differentiation, but the function of Smad4 in Bmp signaling remains elusive. Here, we show that disruption of the Smad4 gene in cardiac progenitors expressing Sfrp5 led to embryonic lethality with hypoplastic heart formation. Although the expression of Nkx2-5 is regulated by Bmp signaling, expression of Nkx2-5 was weakly detected in the mutant heart. However, the nuclear translocation of Nkx2-5 was impaired. Expression of CK2 or PP1, which could alter the phosphorylation status of the NLS of Nkx2-5, was not affected, but Nkx2-5 was found to bind to Smad4 by co-immunoprecipitation experiments. Introduction of Smad4 into cells derived from Smad4 conditional knockout embryonic hearts restored the nuclear localization of Nkx2-5, and exogenous Nkx2-5 failed to translocate into the nucleus of Smad4-depleted fibroblasts. These results suggest that Smad4 plays an essential role in cardiomyocyte differentiation by controlling not only transcription but also the nuclear localization of Nkx2-5.

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

The most striking consequence of a heart attack is the loss of billions of heart muscle cells, alongside damage to the associated vasculature. The lost cardiovascular tissue is replaced by scar formation, which is non-functional and results in pathological remodelling of the heart and ultimately heart failure. It is, therefore, unsurprising that the heart regeneration field has centred efforts to generate new muscle and blood vessels through targeting cardiomyocyte proliferation and angiogenesis following injury. However, combined insights from embryological studies and regenerative models, alongside the adoption of -omics technology, highlight the extensive heterogeneity of cell types within the forming or re-forming heart and the significant crosstalk arising from non-muscle and non-vessel cells. In this review, we focus on the roles of fibroblasts, immune, conduction system, and nervous system cell populations during heart development and we consider the latest evidence supporting a function for these diverse lineages in contributing to regeneration following heart injury. We suggest that the emerging picture of neurologically, immunologically, and electrically coupled cell function calls for a wider-ranging combinatorial approach to heart regeneration.

Annotation. Diseases of the cardiovascular system occupy a leading place in the structure of non-infectious pathology. In the literature, small abnormalities of the development of the heart are described as abnormally located trabeculae or additional chords in the left ventricle of the heart or mitral valve prolapse. At present, the problem of cerebral hemodynamic disorders in children with them is insufficiently studied in medical sources. The aim of our study was to analyze cerebral hemodynamics using ultrasound dopplerography of cerebral vessels in children with abnormally attached chords (AAC) and mitral valve prolapse (MVP). We examined 64 children with AAC and 106 patients with MVP (among whom there were 90 with MVP I degree and 16 with MVP II degree) aged from 13 to 17 years, who formed the main group. The results were compared with the data of the control group, which included 23 almost healthy children also aged 13–17 years. All children underwent ultrasound dopplerography of extra- and intracranial vessels and veins of the brain. Evaluated the average values in the form of M±m. The values of the differences between the indicators of the two samples were evaluated by Student’s parametric criterion (t) using a special program such as Microsoft Excel. In children with MVP I deg. we found a pronounced violation of blood flow in a. vertebralis on both sides with diagnostically significant (р

Cardiac morphogenesis requires an intricate orchestration of mechanical stress to sculpt the heart as it transitions from a straight tube to a multichambered adult heart. Mechanical properties are fundamental to this process, involved in a complex interplay with function, morphology, and mechanotransduction. In the current work, we propose a pressurization technique applied to the zebrafish atrium to quantify mechanical properties of the myocardium under passive tension. By further measuring deformation, we obtain a pressure-stretch relationship that is used to identify constitutive models of the zebrafish embryonic cardiac tissue. Two-dimensional results are compared with a three-dimensional finite element analysis based on reconstructed embryonic heart geometry. Through these steps, we found that the myocardium of zebrafish results in a stiffness on the order of 10 kPa immediately after the looping stage of development. This work enables the ability to determine how these properties change under normal and pathological heart development.

Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinβ, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinβ in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinβ mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinβ knock-out mice—indicating a functional and genetic interaction between Xinβ and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinβ modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases., Intercalated discs ensure mechanical and electrochemical coupling during contraction of the heart. Here, the authors show that loss of Xinβ results in cardiomyocyte proliferation defects and cardiomyopathy by influencing the Hippo-YAP signalling pathway, thus affecting cardiac development and function.

Endocardium is critically important for proper function of the cardiovascular system. Not only does endocardium connect the heart to blood vasculature, it also plays an important role in heart morphogenesis, valve formation, and ventricular trabeculation. The extracellular protein Fibronectin (Fn1) promotes endocardial differentiation, but the signaling pathways downstream of Fn1 that regulate endocardial development are not understood. Here, we analyzed the role of the Fibronectin receptors Integrin alpha5 (Itga5) and Integrin alpha4 (Itga4) in zebrafish heart development. We show that itga5 mRNA is expressed in both endocardium and myocardium during early stages of heart development. Through analysis of both itga5 single mutants and itga4;itga5 double mutants, we show that loss of both itga5 and itga4 results in enhanced defects in endocardial differentiation and morphogenesis compared to loss of itga5 alone. Loss of both itga5 and itga4 results in cardia bifida and severe myocardial morphology defects. Finally, we find that loss of itga5 and itga4 results in abnormally narrow anterior endodermal sheet morphology. Together, our results support a model in which Itga5 and Itga4 cooperate to promote endocardial differentiation, medial migration of endocardial and myocardial cells, and morphogenesis of anterior endoderm.

The aim of this study was to establish a feasible and robust magnetic resonance imaging protocol for the quantitative assessment of cardiac function in marmosets and to present normal values of cardiac function across different ages from young adult, middle-aged, to very old clinically healthy animals. Cardiac MRI of 33 anesthetized marmosets at the age of 2–15 years was performed at 9.4 T using IntraGate-FLASH that operates without any ECG-triggering and breath holding. Normalized to post-mortem heart weight, the left ventricular end-diastolic volume (LV-EDV) was significantly reduced in older marmosets. The LV end-systolic volume (LV-ESV) and the LV stroke volume (LV-SV) showed a similar trend while the LV ejection fraction (LV-EF) and wall thickening remained unchanged. Similar observations were made for the right ventricle. Moreover, the total ventricular myocardial volume was lower in older monkeys while no significant difference in heart weight was found. In conclusion, IntraGate-FLASH allowed for quantification of left ventricular cardiac function but seems to underestimate the volumes of the right ventricle. Although less strong and without significant sex differences, the observed age related changes were similar to previously reported findings in humans supporting marmosets as a model system for age related cardiovascular human diseases.

Cardiac maturation lays the foundation for postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. By integrating single-cell RNA-sequencing data of mouse hearts at multiple postnatal stages, we construct cellular interactomes and regulatory signaling networks. Here we report switching of fibroblast subtypes from a neonatal to adult state and this drives cardiomyocyte maturation. Molecular and functional maturation of neonatal mouse cardiomyocytes and human embryonic stem cell-derived cardiomyocytes are considerably enhanced upon co-culture with corresponding adult cardiac fibroblasts. Further, single-cell analysis of in vivo and in vitro cardiomyocyte maturation trajectories identify highly conserved signaling pathways, pharmacological targeting of which substantially delays cardiomyocyte maturation in postnatal hearts, and markedly enhances cardiomyocyte proliferation and improves cardiac function in infarcted hearts. Together, we identify cardiac fibroblasts as a key constituent in the microenvironment promoting cardiomyocyte maturation, providing insights into how the manipulation of cardiomyocyte maturity may impact on disease development and regeneration., How cardiomyocytes mature and what regulates this is unclear. Here, the authors use single-cell analysis to examine how the population of murine cardiac fibroblasts changes during development and affects maturation of cardiomyocytes.

Background/Aims: Congenital heart disease (CHD) is one of the most common and severe congenital defects. The incidence of fetal cardiac malformation is increased in the context of maternal gestational diabetes mellitus (GDM). Therefore, we wanted to determine whether abnormalities in the insulin signaling pathway are associated with the occurrence of nonsyndromic CHD (ns-CHD). Methods: We used digital gene expression profiling (DGE) of right atrial myocardial tissue samples from eight ns-CHD patients and four controls. The genes potentially associated with CHD were validated by real-time fluorescence quantitative PCR analysis of right atrial myocardial tissues from 37 patients and 10 controls and the H9C2 cell line. Results: The results showed that the insulin signaling pathway, which is mediated by the SHC gene family, was inhibited in the ns-CHD patients. The expression levels of five genes (PTPRF, SHC4, MAP2K2, MKNK2, and ELK1) in the pathway were significantly down-regulated in the patients’ atrial tissues (P

Vorticity is a novel index that reflects diastolic function of left ventricle. The size of the ventricle can influence the ventricular diastolic blood flow. We evaluated effect of ventricular size on diastolic function and diastolic intracardiac blood flow using a particular species of dogs, which has a wide range of body size. Vector flow mapping was used for evaluation of intracardiac blood flow, and intraventricular pressure gradient (IVPG) was used for evaluation of diastolic function. 58 dogs weighing 1.3–42.3 kg were included in this study. Vorticity was found to be inversely proportional to the length of the ventricular chamber. Intraventricular pressure difference was positively correlated with the length of the left ventricle, whereas IVPG was not. This study showed that the vorticity is influenced by the size of the left ventricle independently of other factors. To evaluate the hemodynamic state of each individual appropriately by using vorticity and IVPD, ventricular size should be taken into account especially in the field of veterinary medicine and human pediatric and adolescent cardiology.

Most cell fate trajectories during development follow a diverging, tree-like branching pattern, but the opposite can occur when distinct progenitors contribute to the same cell type. During this convergent differentiation, it is unknown if cells “remember” their origins transcriptionally or whether this influences cell behavior. Most coronary blood vessels of the heart develop from two different progenitor sources—the endocardium (Endo) and sinus venosus (SV)—but whether transcriptional or functional differences related to origin are retained is unknown. We addressed this by combining lineage tracing with single-cell RNA sequencing (scRNAseq) in embryonic and adult mouse hearts. Shortly after coronary development begins, capillary ECs transcriptionally segregated into two states that retained progenitor-specific gene expression. Later in development, when the coronary vasculature is well-established but still remodeling, capillary Ecs again segregated into two populations, but transcriptional differences were primarily related to tissue localization rather than lineage. Specifically, ECs in the heart septum expressed genes indicative of increased local hypoxia and decreased blood flow. Adult capillary ECs were more homogeneous with respect to both lineage and location. In agreement, SV- and Endo-derived ECs in adult hearts displayed similar responses to injury. Finally, scRNAseq of developing human coronary vessels indicated that the human heart followed similar principles. Thus, over the course of development, transcriptional heterogeneity in coronary ECs is first influenced by lineage, then by location, until heterogeneity declines in the homeostatic adult heart. These results highlight the plasticity of ECs during development, and the validity of the mouse as a model for human coronary development.

Sex disparities in cardiac homeostasis and heart disease are well documented, with differences attributed to actions of sex hormones. However, studies have indicated sex chromosomes act outside of the gonads to function without mediation by gonadal hormones. Here, we performed transcriptional and proteomics profiling to define differences between male and female mouse hearts. We demonstrate, contrary to current dogma, cardiac sex disparities are controlled not only by sex hormones but also through a sex-chromosome mechanism. Using Turner syndrome (XO) and Klinefelter (XXY) models, we find the sex-chromosome pathway is established by X-linked gene dosage. We demonstrate cardiac sex disparities occur at the earliest stages of heart formation, a period before gonad formation. Using these datasets, we identify and define a role for alpha-1B-glycoprotein (A1BG), showing loss of A1BG leads to cardiac defects in females, but not males. These studies provide resources for studying sex-biased cardiac disease states.

Background Cardiomyopathy is a kind of cardiovascular diseases, which makes it more difficult for the heart to pump blood to other parts of the body, eventually leading to heart failure. Naoxintong (NXT), as a traditional Chinese Medicine (TCM) preparation, is widely used in the treatment of cardiovascular diseases, including cardiomyopathy, while its underlying mechanism has not been fully elucidated. The purpose of this study is to investigate the therapeutic effect of NXT on cardiomyopathy and its molecular mechanism in zebrafish model. Methods The zebrafish cardiomyopathy model was established using terfenadine (TFD) and treated with NXT. The therapeutic effect of NXT on cardiomyopathy was evaluated by measuring the heart rate, the distance between the sinus venosus and bulbus arteriosus (SV-BA), the pericardial area, and the blood flow velocity of zebrafish. Then, the zebrafish hearts were isolated and collected; transcriptome analysis of NXT on cardiomyopathy was investigated. Moreover, the heg1 mutant of zebrafish congenital cardiomyopathy model was used to further validate the therapeutic effect of NXT on cardiomyopathy. Additionally, UPLC analysis combined with the zebrafish model investigation was performed to identify the bioactive components of NXT. Results In the TFD-induced zebrafish cardiomyopathy model, NXT treatment could significantly restore the cardiovascular malformations caused by cardiac dysfunction. Transcriptome and bioinformatics analyses of the TFD and TFD + NXT treated zebrafish developing hearts revealed that the differentially expressed genes were highly enriched in biological processes such as cardiac muscle contraction and heart development. As a cardiac development protein associated with cardiomyopathy, HEG1 had been identified as one of the important targets of NXT in the treatment of cardiomyopathy. The cardiovascular abnormalities of zebrafish heg1 mutant could be recovered significantly from NXT treatment, including the expanded atrial cavity and blood stagnation. qRT-PCR analysis further showed that NXT could restore cardiomyopathy phenotype in zebrafish through HEG1-CCM signaling. Among the seven components identified in NXT, paeoniflorin (PF) and salvianolic acid B (Sal B) were considered to be the main bioactive ones with myocardial protection. Conclusion NXT presented myocardial protective effect and could restore myocardial injury and cardiac dysfunction in zebrafish; the action mechanism was involved in HEG1-CCM signaling.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) allow investigations in a human cardiac model system, but disorganized mechanics and immaturity of hPSC-CMs on standard two-dimensional surfaces have been hurdles. Here, we developed a platform of micron-scale cardiac muscle bundles to control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips on two-dimensional elastomer substrates with far greater throughput than single cell methods. By defining geometry and workload in this reductionist platform, we show that myofibrillar alignment and auxotonic contractions at physiologic workload drive maturation of contractile function, calcium handling, and electrophysiology. Using transcriptomics, reporter hPSC-CMs, and quantitative immunofluorescence, these cardiac muscle bundles can be used to parse orthogonal cues in early development, including contractile force, calcium load, and metabolic signals. Additionally, the resultant organized biomechanics facilitates automated extraction of contractile kinetics from brightfield microscopy imaging, increasing the accessibility, reproducibility, and throughput of pharmacologic testing and cardiomyopathy disease modeling., Investigations of human cardiac disease involving human pluripotent stem cell-derived cardiomyocytes are limited by the disorganized presentation of biomechanical cues resulting in cell immaturity. Here the authors develop a platform of micron-scale 2D cardiac muscle bundles to precisely deliver physiologic cues, improving reproducibility and throughput.

Summary Congenital heart defects are a feature of several genetic haploinsufficiency syndromes, often involving transcriptional regulators. One property of haploinsufficient genes is their propensity for network interactions at the gene or protein level. In this article we took advantage of an online dataset of high throughput screening of mutations that are embryonic lethal in mice. Our aim was to identify new genes where the loss of function caused cardiovascular phenotypes resembling the 22q11.2 deletion syndrome models, that is, heterozygous and homozygous loss of Tbx1. One gene with a potentially haploinsufficient phenotype was identified, Setd5, thought to be involved in chromatin modification. We found murine Setd5 haploinsufficiency to be associated with double outlet right ventricle and perimembranous ventricular septal defect, although no genetic interaction with Tbx1 was detected. Conditional mutagenesis revealed that Setd5 was required in cardiopharyngeal mesoderm for progression of the heart tube through the ballooning stage to create a four‐chambered heart.

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stagespecific roles during cardiac development.

This work was supported by: Spanish Ministry of Economy, Industry and Competitivity (BFU2017-83907-P to RM-C and RC and PID2019-107492GB-I00 to AA and DF), Consejeria de Salud, Junta de Andalucia (PC0066?2017/PC-0081-2017 to RC, JV, and JG), Instituto de Salud Carlos III-TERCEL network (RD16/0011/0030 to RM-C and RC), Instituto de Salud Carlos III-CIBERCV "Enfermedades Cardiovasculares" (CB16/11/00360 to MJ-N), and Consejeria de Economia y Conocimiento, Junta de Andalucia (UMA18-FEDERJA-146 to RM-C and RC and FEDER-UJA to AA and DF)., Expression of Wilms’ tumor suppressor transcription factor (WT1) in the embryonic epicardium is essential for cardiac development, but its myocardial expression is little known. We have found that WT1 is expressed at low levels in 20–25% of the embryonic cardiomyocytes. Conditional ablation of WT1 using a cardiac troponin T driver (Tnnt2Cre) caused abnormal sinus venosus and atrium development, lack of pectinate muscles, thin ventricular myocardium and, in some cases, interventricular septum and cardiac wall defects, ventricular diverticula and aneurisms. Coronary development was normal and there was not embryonic lethality, although survival of adult mutant mice was reduced probably due to perinatal mortality. Adult mutant mice showed electrocardiographic anomalies, including increased RR and QRS intervals, and decreased PR intervals. RNASeq analysis identified differential expression of 137 genes in the E13.5 mutant heart as compared to controls. GO functional enrichment analysis suggested that both calcium ion regulation and modulation of potassium channels are deeply altered in the mutant myocardium. In summary, together with its essential function in the embryonic epicardium, myocardial WT1 expression is also required for normal cardiac development., Spanish Ministry of Economy, Industry and Competitivity BFU2017-83907-P PID2019-107492GB-I00, Junta de Andalucia PC0066?2017/PC-0081-2017, Instituto de Salud Carlos III-TERCEL network RD16/0011/0030 Instituto de Salud Carlos III-CIBERCV "Enfermedades Cardiovasculares" CB16/11/00360, Junta de Andalucia UMA18-FEDERJA-146

Loss of function of ZIC3 causes heterotaxy (OMIM #306955), a disorder characterized by organ laterality defects including complex heart defects. Studies using Zic3 mutant mice have demonstrated that loss of Zic3 causes heterotaxy due to defects in establishment of left–right (LR) signaling, but the mechanistic basis for these defects remains unknown. Here, we demonstrate Zic3 null mice undergo cilia positioning defects at the embryonic node consistent with impaired planar cell polarity (PCP). Cell-based assays demonstrate that ZIC3 must enter the nucleus to regulate PCP and identify multiple critical ZIC3 domains required for regulation of PCP signaling. Furthermore, we show that Zic3 displays a genetic interaction with the PCP membrane protein Vangl2 and the PCP effector genes Rac1 and Daam1 resulting in increased frequency and severity of neural tube and heart defects. Gene and protein expression analyses indicate that Zic3 null embryos display disrupted expression of PCP components and reduced phosphorylation of the core PCP protein DVL2 at the time of LR axis determination. These results demonstrate that ZIC3 interacts with PCP signaling during early development, identifying a novel role for this transcription factor, and adding additional evidence about the importance of PCP function for normal LR patterning and subsequent heart development.

Background Modeling cardiovascular diseases in mice has provided invaluable insights into the cause of congenital heart disease. However, the small size of the mouse heart has precluded translational studies. Given current high‐efficiency gene editing, congenital heart disease modeling in other species is possible. The pig is advantageous given its cardiac anatomy, physiology, and size are similar to human infants. We profiled pig cardiovascular development and generated genetically edited pigs with congenital heart defects. Methods and Results Pig conceptuses and fetuses were collected spanning 7 stages (day 20 to birth at day 115), with at least 3 embryos analyzed per stage. A combination of magnetic resonance imaging and 3‐dimensional histological reconstructions with episcopic confocal microscopy were conducted. Gross dissections were performed in late‐stage or term fetuses by using sequential segmental analysis of the atrial, ventricular, and arterial segments. At day 20, the heart has looped, forming a common atria and ventricle and an undivided outflow tract. Cardiac morphogenesis progressed rapidly, with atrial and outflow septation evident by day 26 and ventricular septation completed by day 30. The outflow and atrioventricular cushions seen at day 20 undergo remodeling to form mature valves, a process continuing beyond day 42. Genetically edited pigs generated with mutation in chromatin modifier SAP130 exhibited tricuspid dysplasia, with tricuspid atresia associated with early embryonic lethality. Conclusions The major events in pig cardiac morphogenesis are largely complete by day 30. The developmental profile is similar to human and mouse, indicating gene edited pigs may provide new opportunities for preclinical studies focused on outcome improvements for congenital heart disease.

The neural crest (NC) is a multipotent and temporarily migratory cell population stemming from the dorsal neural tube during vertebrate embryogenesis. Cardiac neural crest cells (NCCs), a specified subpopulation of the NC, are vital for normal cardiovascular development, as they significantly contribute to the pharyngeal arch arteries, the developing cardiac outflow tract (OFT), cardiac valves, and interventricular septum. Various signaling pathways are shown to orchestrate the proper migration, compaction, and differentiation of cardiac NCCs during cardiovascular development. Any loss or dysregulation of signaling pathways in cardiac NCCs can lead to abnormal cardiovascular development during embryogenesis, resulting in abnormalities categorized as congenital heart defects (CHDs). This review focuses on the contributions of cardiac NCCs to cardiovascular formation, discusses cardiac defects caused by a disruption of various regulatory factors, and summarizes the role of multiple signaling pathways during embryonic development. A better understanding of the cardiac NC and its vast regulatory network will provide a deeper insight into the mechanisms of the associated abnormalities, leading to potential therapeutic advancements.

The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in WT postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in WT hearts.

MicroRNAs (miRNAs) are translational regulatory molecules with recognised roles in heart development and disease. Therefore, it is important to define the human miRNA expression profile in cardiac progenitors and early-differentiated cardiomyocytes and to determine whether critical cardiac transcription factors such as NKX2-5 regulate miRNA expression. We used an NKX2-5eGFP/w reporter line to isolate both cardiac committed mesoderm and cardiomyocytes. We identified 11 miRNAs that were differentially expressed in NKX2-5 -expressing cardiac mesoderm compared to non-cardiac mesoderm. Subsequent profiling revealed that the canonical myogenic miRNAs including MIR1-1, MIR133A1 and MIR208A were enriched in cardiomyocytes. Strikingly, deletion of NKX2-5 did not result in gross changes in the cardiac miRNA profile, either at committed mesoderm or cardiomyocyte stages. Thus, in early human cardiomyocyte commitment and differentiation, the cardiac myogenic miRNA program is predominantly regulated independently of the highly conserved NKX2-5 -dependant gene regulatory network.

Myocardial damage due to dysfunctional myocardium has been increasing, and the prognosis of pharmacological and device-based therapies remain poor. Isl1-expressing cells were thought to be progenitor cells for cardiomyocyte proliferation after specific stimuli. However, the true origin of the proliferating myocardiac cells and the role of Isl1 in adult mammals remain unresolved. In this study, Isl1-CreERT2 knock-in mouse model was constructed using CRISPR/Cas9 technology. Using tamoxifen-inducible Isl1-CreERT/Rosa26R-LacZ system, Isl1+cells and their progeny were permanently marked by lacZ-expression. X-gal staining, immunostaining, and quantitative PCR were then used to reveal the fate of Isl1+cells under physiological and exercise conditions in mouse hearts from embryonic stage to adulthood. Isl1+cells were found to localize to the sinoatrial node, atrioventricular node, cardiac ganglia, aortic arch, and pulmonary roots in adult mice heart. However, they did not act as cardiac progenitor cells under physiological and exercise conditions. Although Isl1+cells showed progenitor cell properties in early mouse embryos (E7.5), this ability was lost by E9.5. Furthermore, although the proliferation and regeneration of heart cell was observed in response to exercise, the cells associated were not Isl1 positive.

Despite the great progress on the cell biology of programmed cell death (PCD), its incidence and exact time course during embryonic and particular heart development are still unclear. This is also due to the lack of models enabling to directly identify and monitor PCD cells at different time points in vivo. Herein we report generation of transgenic murine embryonic stem cell and mouse models expressing secreted Annexin V-YFP under control of the CAG promoter. This enables to visualize and quantify PCD in vitro and in vivo during embryonic development. At early embryonic stages we found Annexin V-YFP+ fluorescent cells in known areas of PCD, such as the otic ring and at the site of neural tube closing, underscoring its specificity for detection of PCD. We have focused our detailed analysis primarily on PCD in the embryonic heart for a better understanding of its role during development. Our findings reveal that PCD peaks at early stages of cardiogenesis (E9.5–E13.5) and strongly decreases thereafter. Moreover, the PCD cells in the heart are predominantly cardiomyocytes, and an unexpected area of prominent cardiac PCD are the ventricular trabeculae (E9.5–E14.5). Thus, the sA5-YFP mouse line provides novel insight into the incidence and relevance of cardiac PCD during embryonic development ex- and in vivo.

Women with pre‐gestational diabetes have a higher risk of producing children with congenital heart defects (CHDs), caused predominantly by hyperglycemia‐induced oxidative stress. In this study, we evaluated if exercise during pregnancy could mitigate oxidative stress and reduce the incidence of CHDs in the offspring of diabetic mice. Female mice were treated with streptozotocin to induce pre‐gestational diabetes, then mated with healthy males to produce offspring. They were also given access to running wheels 1 week before mating and allowed to exercise voluntarily until E18.5. Heart morphology, gene expression, and oxidative stress were assessed in foetal hearts. Maternal voluntary exercise results in a significantly lower incidence of CHDs from 59.5% to 25%. Additionally, diabetes‐induced defects in coronary artery and capillary morphogenesis were also lower with exercise. Myocardial cell proliferation and epithelial‐mesenchymal transition at E12.5 was significantly lower with pre‐gestational diabetes which was mitigated with maternal exercise. Cardiac gene expression of Notch1, Snail1, Gata4 and Cyclin D1 was significantly higher in the embryos of diabetic mice that exercised compared to the non‐exercised group. Furthermore, maternal exercise produced lower reactive oxygen species (ROS) and oxidative stress in the foetal heart. In conclusion, maternal exercise mitigates ROS and oxidative damage in the foetal heart, and results in a lower incidence of CHDs in the offspring of pre‐gestational diabetes. Exercise may be an effective intervention to compliment clinical management and further minimize CHD risk in mothers with diabetes.

Early embryonic endocardium undergoes endothelial-to-mesenchymal transition to form cardiac cushion mesenchymal cells (MCs). Embryonic endocardium also gives rise to fibroblasts, intramyocardial adipocytes, and coronary mural cells, including smooth muscle cells and pericytes, in development. Whether endocardial cells directly differentiate into fibroblasts, coronary mural cells, and adipocytes or indirectly via an intermediate stage of endocardial-derived cushion MCs remains unknown. In addition to endocardium, epicardium and neural crest also contribute to cardiac cushion MCs. Given the developmental heterogeneity of cushion MCs and the lack of specific markers for endocardial-derived cushion MCs, conventional genetic lineage tracing utilizing Cre recombinase driven by one specific regulatory element is not sufficient to examine the fates of endocardial-derived cushion MCs. Intersectional genetic targeting approaches, which combine regulatory elements from two or more genes, have been employed to increase the specificity of cell targeting. Here, we developed a dual-recombinase intersectional targeting approach using Nfatc1–Dre, Sox9–CreER, and Cre/Dre double-dependent reporter Ai66 to specifically label endocardial-derived cushion MCs. Taking advantage of intersectional lineage tracing, we found that a subset of cardiac cells including fibroblasts, coronary mural cells, and intramyocardial adipocytes in adult hearts were derived from endocardial-derived cushion MCs. Our study suggests that embryonic endocardium contributes to cushion MCs first, and then endocardial-derived cushion MCs migrate into myocardium and differentiate into fibroblasts, coronary mural cells, and adipocytes in development. Understanding developmental origins of cardiac cell lineages will provide us more insights into cardiac development, regeneration, and diseases.

The Drosophila spinster (spin) mutant was isolated as a mutant that showed abnormal morphology and function in the nervous system. The spin defect induces neural degeneration similar to human lysosomal storage diseases. Various studies have shown that Spin proteins are localized in lysosomes and participate in the late stages of the autophagic process. Vertebrates have three spinster orthologs, Spns1, Spns2, and Spns3. A defect in Spns1 caused a short lifespan with aberrant lysosomal function in zebrafish. Spns2 was originally isolated as the gene responsible for abnormal heart development and was identified as a sphingosine 1-phosphate transporter in zebrafish. An endothelial cell-specific defect in Spns2 resulted in impaired egress of lymphocytes and the prevention of tumor metastasis in mice. Herein, I reviewed the history of spin/Spns research and discussed the conserved and newly diverged spin/Spns function and possible implications for human diseases.

There are intense interests in discovering proregenerative medicine leads that can promote cardiac differentiation and regeneration, as well as repair damaged heart tissues. We have combined zebrafish embryo-based screens with cardiomyogenesis assays to discover selective small molecules that modulate heart development and regeneration with minimal adverse effects. Two related compounds with novel structures, named as Cardiomogen 1 and 2 (CDMG1 and CDMG2), were identified for their capacity to promote myocardial hyperplasia through expansion of the cardiac progenitor cell population. We find that Cardiomogen acts as a Wnt inhibitor by targeting β-catenin and reducing Tcf/Lef-mediated transcription in cultured cells. CDMG treatment of amputated zebrafish hearts reduces nuclear β-catenin in injured heart tissue, increases cardiomyocyte (CM) proliferation, and expedites wound healing, thus accelerating cardiac muscle regeneration. Importantly, Cardiomogen can alleviate the functional deterioration of mammalian hearts after myocardial infarction. Injured hearts exposed to CDMG1 display increased newly formed CMs and reduced fibrotic scar tissue, which are in part attributable to the β-catenin reduction. Our findings indicate Cardiomogen as a Wnt inhibitor in enhancing injury-induced CM proliferation and heart regeneration, highlighting the values of embryo-based small molecule screens in discovery of effective and safe medicine leads.

R-loops are naturally occurring transcriptional intermediates containing RNA/DNA hybrids. Excessive R-loops cause genomic instability, DNA damage, and replication stress. Senataxin-associated exonuclease (San1) is a protein that interacts with Senataxin (SETX), a helicase resolving R-loops. It remains unknown if R-loops-induced DNA damage plays a role in the heart, especially in the proliferative neonatal cardiomyocytes (CMs). San1-/- mice were generated using the CRISPR/Cas9 technique. The newborn San1-/- mice show no overt phenotype, but their hearts were smaller with larger, yet fewer CMs. CM proliferation was impaired with reduced cell cycle-related transcripts and proteins. S9.6 staining revealed that excessive R-loops accumulated in the nucleus of neonatal San1-/- CMs. Increased γH2AX staining on newborn and adult heart sections exhibited increased DNA damage. Similarly, San1-/- AC16-cardiomyocytes showed cumulative R-loops and DNA damage, leading to the activation of cell cycle checkpoint kinase ATR and PARP1 hyperactivity, arresting G2/M cell-cycle and CM proliferation. Together, the present study uncovers an essential role of San1 in resolving excessive R-loops that lead to DNA damage and repressing CM proliferation, providing new insights into a novel biological function of San1 in the neonatal heart. San1 may serve as a novel therapeutic target for the treatment of hypoplastic cardiac disorders.

Di-2-ethylhexyl phthalate (DEHP) is a type of plasticizer widely used in industry. It is well-known for its toxic effects to endocrine and reproductive systems and has been detected in amniotic fluid and placenta. In the present study, we explored the effects of DEHP on heart development by using zebrafish as a model organism. DEHP (0.02 pg) was injected into the yolk sac of zebrafish embryos at the one-cell stage. No significant difference was found in embryonic lethality between control and DEHP groups at 1-day postfertilization (dpf), but mortality significantly increased in DEHP groups at 2 and 3 dpf. The average heart rate was significantly reduced in the surviving DEHP-treated zebrafish larvae at 3 and 4 dpf. In addition, massive pericardial edema was found in DEHP-treated zebrafish (12.6 ± 1.5%), which was significantly higher than that of the control group. Serious heart looping disorder was also observed in DEHP-treated larvae, mainly manifested with an elongated atrial-ventricular distance. Moreover, the expression of heart development transcription factors was affected by DEHP injection. Real-time polymerase chain reaction confirmed that five transcription factors ( hand2, tp53, mef2c, esr1, and tbx18) were significantly downregulated in the DEHP group at 2 dpf, and three transcription factors ( zic3, tcf21, and gata4) were significantly upregulated. Our results emphasize the need for the development of a nontoxic plasticizer to prevent possible deleterious effects on humans and other life-forms.

During development a pool of precursors form a heart with atrial and ventricular chambers that exhibit distinct transcriptional and electrophysiological properties. Normal development of these chambers is essential for full term survival of the fetus, and deviations result in congenital heart defects. The large number of genes that may cause congenital heart defects when mutated, and the genetic variability and penetrance of the ensuing phenotypes, reveals a need to understand the molecular mechanisms that allow for the formation of chamber-specific cardiomyocyte differentiation. We find that in the developing zebrafish heart, mRNA for a secreted Semaphorin (Sema), Sema3fb, is expressed by all cardiomyocytes, whereas mRNA for its receptor Plexina3 (Plxna3) is expressed by ventricular cardiomyocytes. In sema3fb CRISPR zebrafish mutants, ventricular chamber development is impaired; the ventricles of mutants are smaller in size than their wild type siblings, apparently because of differences in cell size and not cell numbers, with ventricular cardiomyocytes failing to undergo normal developmental hypertrophy. Analysis of chamber differentiation indicates defects in chamber specific gene expression at the border between the ventricular and atrial chambers, with spillage of ventricular chamber genes into the atrium, and vice versa, and a failure to restrict bmp4a mRNA to the atrioventricular canal. The disrupted atrioventricular border region in mutants is accompanied by hypoplastic heart chambers and impaired cardiac function. These data suggest a model whereby cardiomyocytes secrete a Sema cue that, through spatially restricted expression of the receptor, signals in a ventricular chamber-specific manner to establish a distinct border between atrial and ventricular chambers that is important for functional development of the heart.

International audience; Branchiomeric muscles of the head and neck originate in a population of cranial mesoderm termed cardiopharyngeal mesoderm that also contains progenitor cells contributing to growth of the embryonic heart. Retrospective lineage analysis has shown that branchiomeric muscles share a clonal origin with parts of the heart, indicating the presence of common heart and head muscle progenitor cells in the early embryo. Genetic lineage tracing and functional studies in the mouse, as well as in Ciona and zebrafish, together with recent experiments using single cell transcriptomics and multipotent stem cells, have provided further support for the existence of bipotent head and heart muscle progenitor cells. Current challenges concern defining where and when such common progenitor cells exist in mammalian embryos and how alternative myogenic derivatives emerge in cardiopharyngeal mesoderm. Addressing these questions will provide insights into mechanisms of cell fate acquisition and the evolution of vertebrate musculature, as well as clinical insights into the origins of muscle restricted myopathies and congenital defects affecting craniofacial and cardiac development.

The cardiac neural crest cells (cNCCs) is a transient, migratory cell population that contribute to the formation of major arteries and the septa and valves of the heart. Abnormal development of cNCCs leads to a spectrum of congenital heart defects that mainly affect the outflow region of the hearts. Signaling molecules and transcription factors are the best studied regulatory events controlling cNCC development. In recent years, however, accumulated evidence supports that epigenetic regulation also plays an important role in cNCC development. Here, we summarize the functions of epigenetic regulators during cNCC development as well as cNCC related cardiovascular defects. These factors include ATP-dependent chromatin remodeling factors, histone modifiers and DNA methylation modulators. In many cases, mutations in the genes encoding these factors are known to cause inborn heart diseases. A better understanding of epigenetic regulators, their activities and their roles during heart development will ultimately contribute to the development of new clinical applications for patients with congenital heart disease.

Congenital heart defects are the most common birth defect and have a clear genetic component, yet genomic structural variations or gene mutations account for only a third of the cases. Epigenomic dynamics during human heart organogenesis thus may play a critical role in regulating heart development. However, it is unclear how histone mark H3K36me3 acts on heart development. Here we report that histone-lysine N-methyltransferase SETD2, an H3K36me3 methyltransferase, is a crucial regulator of the mouse heart epigenome.Setd2is highly expressed in embryonic stages and accounts for a predominate role of H3K36me3 in the heart. Loss ofSetd2in cardiac progenitors results in obvious coronary vascular defects and ventricular non-compaction, leading to fetus lethality in mid-gestation, without affecting peripheral blood vessel, yolk sac, and placenta formation. Furthermore, deletion ofSetd2dramatically decreased H3K36me3 level and impacted the transcriptional landscape of key cardiac-related genes, includingRspo3andFlrt2. Taken together, our results strongly suggest that SETD2 plays a primary role in H3K36me3 and is critical for coronary vascular formation and heart development in mice.

Isoflucypram belongs to the new generation of succinate dehydrogenase inhibitor (SDHI) fungicides that are commonly used in crop fungal disease control. Evidence indicates that isoflucypram poses a potential risk to aquatic organisms. However, the effects of isoflucypram during early embryogenesis are not fully understood. In the present study, zebrafish embryos were exposed to 0.025, 0.25, or 2.5 μM isoflucypram for three days. Isoflucypram caused severe developmental abnormalities (yolk sac edema, pericardial edema, and blood clotting clustering), hatching delay, and decreased heart rates in zebrafish. The expression levels of cardiac-specific genes (nkx2.5, myh7, myl7, and myh6) and erythropoiesis-related genes (gata1a, hbbe1, hbbe2, and alas2) were disrupted after isoflucypram exposure. Furthermore, enrichment analysis indicated that most of the differentially expressed genes (DEGs) were enriched in heart development or hemopoiesis processes. Overall, these findings suggest that exposure to isoflucypram is associated with developmental and cardiovascular toxicity in zebrafish.

Cardiomyocyte (CM) maturation is the transformation of differentiated fetal CMs into adult CMs that involves changes in morphology, cell function and metabolism, and the transcriptome. This process is, however, incomplete and ultimately arrested in pluripotent stem cell-derived CMs (PSC-CMs) in culture, which hinders their broad biomedical application. For this reason, enormous efforts are currently being made with the goal of generating mature PSC-CMs. In this review, we summarize key aspects of maturation observed in native CMs and discuss recent findings on the factors and mechanisms that regulate the process. Particular emphasis is put on transcriptional regulation and single-cell RNA-sequencing analysis that has emerged as a key tool to study time-series gene regulation and to determine the maturation state. We then discuss different biomimetic strategies to enhance PSC-CM maturation and discuss their effects at the single cell transcriptomic and functional levels.

The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF) emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling usingdrl-driven CreERT2 recombinase with a myocardium-specific,myl7-controlledloxPreporter results in predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary.

During early vertebrate heart development, the heart transitions from a linear tube to a complex asymmetric structure. This process includes looping of the tube and ballooning of the emerging cardiac chambers, which occur simultaneously with growth of the heart. A key driver of cardiac growth is deployment of cells from the Second Heart Field (SHF) into both poles of the heart, with cardiac morphogenesis and growth intimately linked in heart development. Laminin is a core component of extracellular matrix (ECM) basement membranes, and although mutations in specific laminin subunits are linked with a variety of cardiac abnormalities, including congenital heart disease and dilated cardiomyopathy, no role for laminin has been identified in early vertebrate heart morphogenesis. We identified dynamic, tissue-specific expression of laminin subunit genes in the developing zebrafish heart, supporting a role for laminins in heart morphogenesis.lamb1amutants exhibit cardiomegaly from 2dpf onwards, with subsequent progressive defects in cardiac morphogenesis characterised by a failure of the chambers to compact around the developing atrioventricular canal. We show that loss oflamb1aresults in excess addition of SHF cells to the atrium, revealing that Lamb1a functions to limit heart size during cardiac development by restricting SHF addition to the venous pole.lamb1amutants exhibit hallmarks of altered haemodynamics, and specifically blocking cardiac contractility inlamb1amutants rescues heart size and atrial SHF addition. Furthermore, we identify that FGF and RA signalling, two conserved pathways promoting SHF addition, are regulated by heart contractility and are dysregulated inlamb1amutants, suggesting that laminin mediates interactions between SHF deployment, heart biomechanics, and biochemical signalling during heart development. Together, this describes the first requirement for laminins in early vertebrate heart morphogenesis, reinforcing the importance of specialised ECM composition in cardiac development.

Children with congenital heart defects (CHDs) have increased risk of cognitive disabilities for reasons not fully understood. Previous studies have indicated signs of disrupted fetal brain growth from mid-gestation measured with ultrasound and magnetic resonance imaging (MRI) and infants with CHDs have decreased brain volumes at birth. We measured the total and regional brain volumes of infants with and without CHDs using MRI to investigate, if certain areas of the brain are at particular risk of disrupted growth. MRI brain volumetry analyses were performed on 20 infants; 10 with- (postmenstrual age 39–54 weeks, mean 44 weeks + 5 days) and 10 without CHDs (postmenstrual age 39–52 weeks, mean 43 weeks + 5 days). In six infants with- and eight infants without CHDs grey and white matter were also differentiated. Infants with CHDs had smaller brains (48 ml smaller; 95% CI, 6.1–90; p = 0.03), cerebrums (37.8 ml smaller; 95% CI, 0.8–74.8; p = 0.04), and cerebral grey matter (25.8 ml smaller; 95% CI, 3.5–48; p = 0.03) than infants without CHD. Brain volume differences observed within weeks after birth in children with CHDs confirm that the brain impact, which increase the risk of cognitive disabilities, may begin during pregnancy.

Among the three transforming growth factor beta (TGFβ) ligands, TGFβ2 is essential for heart development and is produced by multiple cell types, including myocardium. Heterozygous mutations in TGFB2 in patients of connective tissue disorders result in congenital heart defects and adult valve malformations, including mitral valve prolapse (MVP) with or without regurgitation. Tgfb2 germline knockout fetuses exhibit multiple cardiac defects but the role of myocardial-TGFβ2 in heart development is yet to be elucidated. Here, myocardial Tgfb2 conditional knockout (CKO) embryos were generated by crossing Tgfb2flox mice with Tgfb2+/−, cTntCre mice. Tgfb2flox/− embryos were normal, viable. Cell fate mapping was done using dual-fluorescent mT/mG+/− mice. Cre-mediated Tgfb2 deletion was assessed by genomic PCR. RNAscope in situ hybridization was used to detect the loss of myocardial Tgfb2 expression. Histological, morphometric, immunohistochemical, and in situ hybridization analyses of CKOs and littermate controls at different stages of heart development (E12.5–E18.5) were used to determine the role of myocardium-derived TGFβ2 in atrioventricular (AV) cushion remodeling and myocardial development. CKOs exhibit a thin ventricular myocardium, AV cushion remodeling defects and developed incomplete AV septation defects. The loss of myocardial Tgfb2 resulted in impaired cushion maturation and dysregulated cell death. Phosphorylated SMAD2, a surrogate for TGFβ signaling, was “paradoxically” increased in both AV cushion mesenchyme and ventricular myocardium in the CKOs. Our results indicate that TGFβ2 produced by cardiomyocytes acting as cells autonomously on myocardium and via paracrine signaling on AV cushions are required for heart development.

Cardiac looping and trabeculation are key processes during cardiac chamber maturation. However, the underlying mechanisms remain incompletely understood. Here, we report the isolation, cloning and characterization of the proprotein convertase furina from the cardiovascular mutant loft in zebrafish. loft is an ethylnitrosourea-induced mutant and has evident defects in the cardiac outflow tract, heart looping and trabeculation, the craniofacial region and pharyngeal arch arteries. Positional cloning revealed that furina mRNA was barely detectable in loft mutants, and loft failed to complement the TALEN-induced furina mutant pku338, confirming that furina is responsible for the loft mutant phenotypes. Mechanistic studies demonstrated that Notch reporter Tg(tp1:mCherry) signals were largely eliminated in mutant hearts, and overexpression of the Notch intracellular domain partially rescued the mutant phenotypes, probably due to the lack of Furina-mediated cleavage processing of Notch1b proteins, the only Notch receptor expressed in the heart. Together, our data suggest a potential post-translational modification of Notch1b proteins via the proprotein convertase Furina in the heart, and unveil the function of the Furina-Notch1b axis in cardiac looping and trabeculation in zebrafish, and possibly in other organisms.