Search

Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A) drive clonal hematopoiesis of indeterminate potential (CHIP) and are associated with adverse prognosis in patients with heart failure (HF). The interactions between CHIP-mutated cells and other cardiac cell types remain unknown.Here, we identify fibroblasts as potential interaction partners of CHIP-mutated monocytes using combined transcriptomic data from peripheral blood mononuclear cells of HF patients with and without CHIP and the cardiac tissue. We demonstrate that CHIP augments macrophage-to-cardiac fibroblasts interactions. Mechanistically, the secretome ofDNMT3A-silenced monocytes leads to myofibroblast activation, partially through epidermal growth factor (EGFR) signaling. Harboring DNMT3A CHIP-driver mutations is associated with increased cardiac interstitial fibrosis in mice and patients, and, thereby, may contribute to the poor outcome.These findings not only identify a novel pathway of DNMT3A CHIP-driver mutation-induced instigation and progression of HF, but may also provide a rationale for the development of new anti-fibrotic strategies.Graphical abstract

Cardiac homeostasis relies on the appropriate provision of nutrients and functional specialization of local endothelial cells. Previously we reported in this journal that the endothelial Eph-ephrin signalling, in particular the ligand EphB4, is required for the maintenance of vascular integrity and correct fatty acid transport uptake in the heart via regulating the caveolar trafficking of the fatty acid receptor CD36. In the mouse, endothelial specific loss-of-function of the receptor EphB4, or its ligand ephrin-B2, induces Dilated Cardiomyopathy (DCM) like defects (Luxán et al., 2019).Here, we have identified new rareEPHB4variants in a cohort of 573 DCM patients. Similar to what we had observed in the EphB4 mutant mice,EPHB4variants carrying patients show an altered expression pattern of CD36 and CAV1 in the myocardium.Our study confirms a crucial role of the Eph-ephrin signalling pathway, and in particular the receptor EPHB4, in the development of DCM in humans.

Aging is a major risk factor for impaired cardiovascular health. The aging myocardium is characterized by electrophysiological dysfunctions such as a reduced heart rate variability. These alterations can be intrinsic within cardiomyocytes, but might be modulated by the cardiac autonomic nervous system, as well1. It is known that nerves align with vessels during development2, but the impact of aging on the cardiac neuro-vascular interface is unknown. Here, we report that aging reduces nerve density specifically in the left ventricle and dysregulates vascular-derived neuro-regulatory genes. Aging leads further to a down-regulation of miR-145 and de-repression of the neuro-repulsive factor Semaphorin-3A. miR-145 deletion increased Sema3a expression and reduced axon density, thus mimicking the observed aged heart phenotype. Removal of senescent cells, which accumulated with chronological age while nerve density declined, rescued from age-induced dennervation, reduced Sema3a expression and preserved heart rate variability. These data suggest that senescence-associated regulation of neuro-regulatory genes contributes to a declined nerve density of the aging heart and thereby to a reduced heart rate variability.

It is well established that the vasculature plays a crucial role in maintaining oxygen and nutrients supply to the heart. Increasing evidence further suggests that the microcirculation has additional roles in supporting a healthy microenvironment. Heart failure is well known to be associated with changes and functional impairment of the microvasculature. The specific ablation of protective signals in endothelial cells in experimental models is sufficient to induce heart failure. Therefore, restoring a healthy endothelium and microcirculation may be a valuable therapeutic strategy to treat heart failure. This review article will summarize the current understanding of the vascular contribution to heart failure with reduced or preserved ejection fraction. Novel therapeutic approaches including next generation pro-angiogenic therapies and non-coding RNA therapeutics, as well as the targeting of metabolites or metabolic signalling, vascular inflammation and senescence will be discussed.

Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.

Cardiac fibroblasts constitute a major cell population in the heart. They secrete extracellular matrix components and various other factors shaping the microenvironment of the heart. In silico analysis of intercellular communication based on single-cell RNA sequencing revealed that fibroblasts are the source of the majority of outgoing signals to other cell types. This observation suggests that fibroblasts play key roles in orchestrating cellular interactions that maintain organ homeostasis but that can also contribute to disease states. Here, we will review the current knowledge of fibroblast interactions in the healthy, diseased, and aging heart. We focus on the interactions that fibroblasts establish with other cells of the heart, specifically cardiomyocytes, endothelial cells and immune cells, and particularly those relying on paracrine, electrical, and exosomal communication modes.

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00882-8.

After myocardial infarct (MI), followed by ischemia and scar formation, interstitial cells play key roles in the adaptation to injury. Endothelial cells (ECs), for instance, can clonally expand, migrate into the infarct area and facilitate crucial functions promoting revascularization, reestablishment of oxygen supply and secretion of paracrine factors. Moreover, ECs can transiently undergo changes towards a mesenchymal phenotype (Endothelial-to-mesenchymal transition; EndMT). Whether this process contributes to long-term cardiac fibrosis or helps to facilitate post-ischemic vessel growth remains controversial. Here, we aim to delineate kinetics and characteristics of phenotypic changes in ECs with single cell RNA-sequencing (scRNA-seq). We performed a time course (homeostasis or 0 day (d), 1d, 3d, 5d, 7d, 14d, 28d post-MI) in mice and isolated the non-cardiomyocyte fraction for scRNA-seq (n=35,312 cells). Pecam1/Cdh5 double positive ECs showed expression of apoptosis, hypoxia and inflammation markers at 3d. Bioinformatic cell cycle analysis predicted high association with proliferative capacities at 3d, indicative of EC turnover post-MI. Metabolism, recently linked to regulate EndMT, was altered. We found genes of the glycolysis and the TCA-cycle pathway upregulated at 1d to 3d, and a decrease of fatty acid signaling genes. At 3d, mesenchymal markers Fn1, Vim, S100a4, Serpine1 transiently increased compared to homeostasis (>1.6-fold, p We additionally used Cdh5-CreERT2; R26-mT/mG mice followed by scRNA-seq to trace the fate of ECs. Bioinformatic analysis of GFP-positive ECs confirmed the gain in mesenchymal marker but revealed no full transition to the mesenchymal state at later timepoints. This suggests a transient mesenchymal activation of ECs rather than a complete lineage transition. We further induced EndMT with TGF-β2 in ECs in vitro and observed reversibility of the phenotype after withdrawal of the stimulus. After treatment, ECs upregulated various mesenchymal marker genes. Withdrawal of TGF-β2 at 3d or 7d, reverted expression to baseline levels. We further determined DNA methylation of EndMT gene loci to assess if TGF-β2 leads to a true fate change but did not observe changes after TGF-β2 stimulation and withdrawal. Taken together, our data suggests that ECs undergo a transient mesenchymal activation concomitant with a metabolic adaptation early after MI but do not acquire a long-term mesenchymal fate. This activation may facilitate EC migration and clonal expansion to regenerate the vascular network. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Center of Cardiovascular Research (DZHK), Deutsche Forschungsgemeinschaft (DFG) CRC1366 Project B4

Introduction The interface between heart and bone emerges as a key trigger of post-infarction inflammation and progression of chronic heart failure (CHF). However, our knowledge on the underlying mechanisms of this interaction is incomplete. Bone vasculature, specifically so-called H-type (Endomucinhigh) endothelial cells (EC), plays a crucial role in maintenance of the bone integrity and regulation of hematopoietic stem cells (HSC). While previous studies in mice showed the reduction of H-type vessels by aging, the impact of ischemic heart disease is unclear. Therefore, we aimed to investigate the effects of myocardial infarction (MI) and chronic heart failure on the vascular bone cell composition in mice and humans. Methods and results Flow cytometric analysis of harvested bones at the different timepoints after MI induction in mice revealed a gradual loss of H-type endothelial cells in the time-course of developing chronic heart failure (P Conclusions Our data show for the first time an impact of myocardial infarction and chronic heart failure on the bone marrow vasculature. These changes seem to be strongly associated with inflammatory response in H-type vessels, which precedes its loss after MI. Specifically, the induction of the inflammatory cytokine IL1b may contribute to the disturbed phenotype. This suggest that inhibition of IL1b (e.g. by canakinumab) be used as a novel strategy to prevent or reverse the deterioration of the vascular bone function in ischemic heart disease. Funding Acknowledgement Type of funding source: None

Aims Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection. Methods and results Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-COV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with COVID-19. Infection of iPS-CMs was dependent on cathepsins and angiotensin-converting enzyme 2 (ACE2), and was blocked by remdesivir. Conclusions This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an ACE2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir. Translational Perspective Although this study cannot address whether cardiac injury and dysfunction in COVID-19 patients is caused by direct infection of cardiomyocytes, the demonstration of direct cardiotoxicity in cardiomyocytes, organ mimics, human heart slices and human hearts warrants the further monitoring of cardiotoxic effects in COVID-19 patients., Graphical Abstract Graphical Abstract

BackgroundThe coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality in COVID-19 patients. It is unclear whether cardiac injury may have been caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here we investigate whether human cardiomyocytes are permissive for SARS-CoV-2 infection. MethodsInfection was induced by two strains of SARS-CoV-2 (FFM1 and FFM2) in human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CM) and in two models of human cardiac tissue. ResultsWe show that SARS-CoV-2 infects hiPS-CM as demonstrated by detection of intracellular double strand viral RNA and viral spike glycoprotein protein expression. Increasing concentrations of virus RNA are detected in supernatants of infected cardiomyocytes, which induced infections in CaCo-2 cell lines documenting productive infections. SARS-COV-2 infection induced cytotoxic and pro-apoptotic effects and abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signaling, apoptosis and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a iPS-derived human 3D cardiosphere tissue models. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. ConclusionsThe demonstration that cardiomyocytes are permissive for SARS-CoV-2 infection in vitro warrants the further in depth monitoring of cardiotoxic effects in COVID-19 patients. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIThis study demonstrates that human cardiac myocytes are permissive for SARS-CoV-2 infection. C_LIO_LIThe study documents that SARS-CoV-2 undergoes a full replicatory circle and induces a cytotoxic response in cardiomyocytes. C_LIO_LIInfection was confirmed in two cardiac tissue models, including living human heart slices. C_LI What are the Clinical Implications?O_LIThe study may provide a rational to explain part of the cardiotoxicity observed in COVID-19 patients C_LIO_LIThe demonstration of direct cardiotoxicity induced by SARS-CoV-2 warrants an in depth further analysis of cardiac tissue of COVID-19 patients and a close monitoring for putative direct cardiomyocyte injury. C_LIO_LIThe established models can be used to test novel therapeutic approaches targeting COVID-19. C_LI

SummaryBone vasculature provides protection and signals necessary to control stem cell quiescence and renewal1. Specifically, type H capillaries, which highly express Endomucin, constitute the endothelial niche supporting a microenvironment of osteoprogenitors and long-term hematopoietic stem cells2–4. The age-dependent decline in type H endothelial cells was shown to be associated with bone dysregulation and accumulation of hematopoietic stem cells, which display cell-intrinsic alterations and reduced functionality3. The regulation of bone vasculature by chronic diseases, such as heart failure is unknown. Here, we describe the effects of myocardial infarction and post-infarction heart failure on the vascular bone cell composition. We demonstrate an age-independent loss of type H bone endothelium in heart failure after myocardial infarction in both mice and in humans. Using single-cell RNA sequencing, we delineate the transcriptional heterogeneity of human bone marrow endothelium showing increased expression of inflammatory genes, including IL1B and MYC, in ischemic heart failure. Inhibition of NLRP3-dependent IL-1β production partially prevents the post-myocardial infarction loss of type H vasculature in mice. These results provide a rationale for using anti-inflammatory therapies to prevent or reverse the deterioration of vascular bone function in ischemic heart disease.

The homeostasis of heart and other organs relies on the appropriate provision of nutrients and functional specialization of the local vasculature. Here, we have used mouse genetics, imaging and cell biology approaches to investigate how homeostasis in the adult heart is controlled by endothelial EphB4 and its ligand ephrin-B2, which are known regulators of vascular morphogenesis and arteriovenous differentiation during development. We show that inducible and endothelial cell-specific inactivation of Ephb4 in adult mice is compatible with survival, but leads to rupturing of cardiac capillaries, cardiomyocyte hypertrophy, and pathological cardiac remodeling. In contrast, EphB4 is not required for integrity and homeostasis of capillaries in skeletal muscle. Our analysis of mutant mice and cultured endothelial cells shows that EphB4 controls the function of caveolae, cell-cell adhesion under mechanical stress and lipid transport. We propose that EphB4 maintains critical functional properties of the adult cardiac vasculature and thereby prevents dilated cardiomyopathy-like defects.

Rationale: The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial–mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. Objective: The aim of this study is to determine the functional specificity of Notch in valve development. Methods and Results: Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial–mesenchymal transition. Mice lacking endocardial Jag1 , Notch1 , or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post–epithelial–mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non–cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1 -mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf , was markedly reduced in Jag1 -mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1 -mutant outflow tract explant cultures rescued the hyperproliferative phenotype. Conclusions: During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial–mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function.

In this chapter we illustrate the signalling interactions of the endocardium with the other cardiac tissues to coordinate cardiac development. First, we describe the developmental origins of the endocardium. Then we focus on the Notch pathway because of its unique signalling activity in the endocardium, and briefly describe the elements of this signalling mechanism and the key cardiogenic processes that require endocardial Notch signalling: patterning of the early embryonic endocardium into prospective territories for valves and ventricular chambers, early valve formation, ventricular trabeculation, and compaction. Finally, we discuss how Notch dysfunction in the endocardium results in cardiac structural malformations that can lead to congenital heart disease.

Introduction Left ventricular noncompaction (LVNC) is characterised by prominent ventricular myocardial trabeculations, composed of sheets of cardiomyocytes. They form early during cardiogenesis but their development is complex. Measuring trabecular complexity in animal models of cardiac disease is important as abnormal trabecular patterns are increasingly been recognised to coexist with several other cardiac conditions, not just LVNC. We describe an innovative approach that utilises fractal algorithms and high-resolution episcopic microscopy (HREM) to study the developmental timing of myocardial trabeculation in mouse and we validate it using a recently described LVNC mouse model (NOTCH pathway regulator Mib1 mutant). Methods HREM (2–3 μm resolution) analysis was performed prospectively on 123 embryonic mouse hearts consisting of wild-type (WT) NIMR:Parkes, WT C57BL/6 and Mib1 flox ; cTnT-cre mutant and WT littermates. HREM permits the 2D/3D imaging of tissue samples as they are physically sectioned. Datasets underwent fractal analysis using a box-counting approach (Figure 1). Results LV trabecular complexity showed a significant drop between E14.5 and E18.5 (Figure 2). Across all embryonic stages, the apical half of the LV retained the highest fractal dimensions (FD) when compared to the base. By E18.5 the myocardium was almost fully compacted registering the lowest FD. For the first time, we demonstrate that strain-specific differences in LV trabecular patterning exist in mouse because NIMR:Parkes compacts earlier than C57BL/6 (Figure 3). Reslicing experiments (Figure 4) and separate validation tests on Mib1 mutants and WT littermates (Fig.5) confirmed how the proposed methodology is a reliable and effective tool for the detection of mutagenesis-related differences in trabeculation. Conclusion Reported here is a method in which sequential, 2D sections of mouse embryo hearts may be analysed using a fractal algorithm to calculate ventricular trabecular complexity – a technique so sensitive, that small inter-strain differences in somitogenesis are detectable in mouse pups. Precise knowledge of the trabecular architecture as it presents itself in WT, is a prerequisite for the correct identification of pathological trabecular phenotypes in mouse models of cardiac disease, explaining the need for a quantitative fractal atlas of trabecular development. Fractal mathematics in combination with HREM has the potential to answer to many developmental biology questions in the heart, with future applicability to other organ systems and to other species.

Left ventricular noncompaction (LVNC) causes prominent ventricular trabeculations and reduces cardiac systolic function. The clinical presentation of LVNC ranges from asymptomatic to heart failure. We show that germline mutations in human MIB1 (mindbomb homolog 1), which encodes an E3 ubiquitin ligase that promotes endocytosis of the NOTCH ligands DELTA and JAGGED, cause LVNC in autosomal-dominant pedigrees, with affected individuals showing reduced NOTCH1 activity and reduced expression of target genes. Functional studies in cells and zebrafish embryos and in silico modeling indicate that MIB1 functions as a dimer, which is disrupted by the human mutations. Targeted inactivation of Mib1 in mouse myocardium causes LVNC, a phenotype mimicked by inactivation of myocardial Jagged1 or endocardial Notch1. Myocardial Mib1 mutants show reduced ventricular Notch1 activity, expansion of compact myocardium to proliferative, immature trabeculae and abnormal expression of cardiac development and disease genes. These results implicate NOTCH signaling in LVNC and indicate that MIB1 mutations arrest chamber myocardium development, preventing trabecular maturation and compaction. 28.054 JCR (2013) Q1, 2/291 Biochemistry & molecular biology, 3/185 Cell biology, 1/122 Medicine, research & experimental

The vertebrate heart is the first organ to form and function during embryogenesis. Primitive streak-derived cardiac progenitors located bilaterally move rostral to form the primitive heart tube that subsequently undergoes rightward looping, remodelling and septation to give rise to the mature four-chambered heart. Tightly regulated tissue interactions orchestrate the patterning, proliferation and differentiation processes that give rise to the adult ventricles. Studies in animal models have demonstrated the crucial function of the Notch signalling pathway in ventricular development and how alterations in human NOTCH signalling may lead to disease in the form of cardiomyopathies, such as left ventricular noncompaction (LVNC). In this review, we discuss how during trabecular formation and ventricular compaction, Dll4-Notch1 signals from chamber endocardium to regulate cardiomyocyte proliferation and differentiation in a noncell autonomous fashion and how, at later stages, myocardial Jag1 and Jag2 activate Notch1 in chamber endocardium to sustain chamber patterning and compaction with simultaneous coronary vessel development mediated by Dll4-Notch1. We suggest that alterations in these molecular mechanisms underlie MIB1-related familial LVNC and favour the hypothesis that this cardiomyopathy has a congenital nature.

The Notch signaling pathway is an ancient and highly conserved signaling pathway that controls cell fate specification and tissue patterning in the embryo and in the adult. Region-specific endocardial Notch activity regulates heart morphogenesis through the interaction with multiple myocardial-, epicardial-, and neural crest–derived signals. Mutations in NOTCH signaling elements cause congenital heart disease in humans and mice, demonstrating its essential role in cardiac development. Studies in model systems have provided mechanistic understanding of Notch function in cardiac development, congenital heart disease, and heart regeneration. Notch patterns the embryonic endocardium into prospective territories for valve and chamber formation, and later regulates the signaling processes leading to outflow tract and valve morphogenesis and ventricular trabeculae compaction. Alterations in NOTCH signaling in the endocardium result in congenital structural malformations that can lead to disease in the neonate and adult heart.

Formation of trabeculae in the embryonic heart and the remodelling that occurs prior to birth is a conspicuous, but poorly understood, feature of vertebrate cardiogenesis. Mutations disrupting trabecular development in the mouse are frequently embryonic lethal, testifying to the importance of the trabeculae, and aberrant trabecular structure is associated with several human cardiac pathologies. Here, trabecular architecture in the developing mouse embryo has been analysed using high-resolution episcopic microscopy (HREM) and three-dimensional (3D) modelling. This study shows that at all stages from mid-gestation to birth, the ventricular trabeculae comprise a complex meshwork of myocardial strands. Such an arrangement defies conventional methods of measurement, and an approach based upon fractal algorithms has been used to provide an objective measure of trabecular complexity. The extent of trabeculation as it changes along the length of left and right ventricles has been quantified, and the changes that occur from formation of the four-chambered heart until shortly before birth have been mapped. This approach not only measures qualitative features evident from visual inspection of 3D models, but also detects subtle, consistent and regionally localised differences that distinguish each ventricle and its developmental stage. Finally, the combination of HREM imaging and fractal analysis has been applied to analyse changes in embryonic heart structure in a genetic mouse model in which trabeculation is deranged. It is shown that myocardial deletion of the Notch pathway component Mib1 (Mib1(flox/flox); cTnT-cre) results in a complex array of abnormalities affecting trabeculae and other parts of the heart. This work was supported by funding to TJM from the Medical Research Council (U117562103); to JCM by the Higher Education Funding Council for England; to JLdlP and GL by grants SAF2010-17555 and SAF2013-45543-R from the Spanish Ministry of Economy and Competitiveness (MINECO); and to GC by a research fellowship at the University College London Biomedical Research Centre from the UK National Institutes of Health Research Cardiometabolic Programme. Sí

The heart is the first organ to develop in the embryo, and its formation is an exquisitely regulated process. Inherited mutations in genes required for cardiac development may cause congenital heart disease (CHD), manifested in the newborn or in the adult. Notch is an ancient, highly conserved signaling pathway that communicates adjacent cells to regulate cell fate specification, differentiation, and tissue patterning. Mutations in Notch signaling elements result in cardiac abnormalities in mice and humans, demonstrating an essential role for Notch in heart development. Recent work has shown that endocardial Notch activity orchestrates the early events as well as the patterning and morphogenesis of the ventricular chambers in the mouse and that inactivating mutations in the NOTCH pathway regulator MIND BOMB-1 (MIB1) cause left ventricular non-compaction (LVNC), a cardiomyopathy of poorly understood etiology. Here, we review these data that shed some light on the etiology of LVNC that at least in the case of that caused by MIB1 mutations has a developmental basis. J.L. de la Pompa is funded by grants SAF2013-45543, RD12/0042/0005 (RIC), and RD12/0019/0003 (TERCEL) from the Spanish Ministry of Economy and Competitiveness (MINECO) and grant FP7-ITN 215761 (NotchIT) from the European Commission. G. Luxan had a PhD fellowship from the MINECO (FPI Program, ref. BES-2008-002904) and G. D’Amato holds a PhD fellowship associated with grant FP7-ITN 215761 (NotchIT). The CNIC is supported by the MINECO and the Pro-CNIC Foundation. Sí

Ventricular chambers are essential for the rhythmic contraction and relaxation occurring in every heartbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is poorly understood. We show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation, and later coordinates ventricular patterning and compaction with coronary vessel development to generate the mature chamber, through a temporal sequence of ligand signalling determined by the glycosyltransferase manic fringe (MFng). Early endocardial expression of MFng promotes Dll4-Notch1 signalling, which induces trabeculation in the developing ventricle. Ventricular maturation and compaction require MFng and Dll4 downregulation in the endocardium, which allows myocardial Jag1 and Jag2 signalling to Notch1 in this tissue. Perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies.

Introduction Dysregulated NOTCH receptor activity has been implicated in breast cancer but the mechanisms by which NOTCH contributes to transformation are not yet clear, as it has context-dependent effects on the properties of transformed cells. Methods We have used various in vitro and in vivo carcinogenic models to analyze the impact of Notch signaling in the onset and progression of breast tumors. Results We found that ectopic expression of the Notch1 intracellular domain (N1ICD) in MCF-7 breast adenocarcinoma cell line caused reduction and delocalization of E-CADHERIN levels and increased migratory and invasive abilities. Notch inhibition in the invasive breast cancer cell line MDA-MB-231 resulted in increased E-CADHERIN expression and a parallel reduction in their invasive capacity. The growth of subcutaneous xenografts produced with MCF-7 cells was boosted after N1ICD induction, in a cell autonomous manner. In vivo Notch1 activation in the mammary gland using the MMTV-Cre driver caused the formation of papillary tumors that showed increased Hes1 and Hey1 expression and delocalized E-cadherin staining. Conclusions These results confirm NOTCH1 as a signal triggering epithelial-mesenchymal transition in epithelial cancer cells, which may have implications in tumor dissemination, metastasis and proliferation in vivo. The identification of specific factors interacting with NOTCH signaling could thus be relevant to fully understanding the role of NOTCH in breast neoplasia., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) or Comunidad de Madrid (CM). The grants were: SAF2010-21205 (MINECO) and MITICP2010/BMD-2502 (CM) to CMA, SAF2011-24453 (MINECO) and INMUNOTHERCAN S2010/BMD-2326 (CM) to SM and SAF2007-62445 and SAF2010-17555 (MINECO) to JLdlP. The CNIC is supported by the MINECO and the Pro-CNIC Foundation.

Cardiac valve formation is crucial for embryonic and adult heart function. Valve malformations constitute the most common congenital cardiac defect, but little is known about the molecular mechanisms regulating valve formation and homeostasis. Here, we show that endocardial Notch1 and myocardial Bmp2 signal integration establish a valve-forming field between 2 chamber developmental domains. Patterning occurs through the activation of endocardial epithelial-to-mesenchymal transition (EMT) exclusively in prospective valve territories. Mice with constitutive endocardial Notch1 activity ectopically express Hey1 and Heyl. They also display an activated mesenchymal gene program in ventricles and a partial (noninvasive) EMT in vitro that becomes invasive upon BMP2 treatment. Snail1, TGF-β2, or Notch1 inhibition reduces BMP2-induced ventricular transformation and invasion, whereas BMP2 treatment inhibits endothelial Gsk3β, stabilizing Snail1 and promoting invasiveness. Integration of Notch and Bmp2 signals is consistent with Notch1 signaling being attenuated after myocardial Bmp2 deletion. Notch1 activation in myocardium extends Hey1 expression to nonchamber myocardium, represses Bmp2, and impairs EMT. In contrast, Notch deletion abrogates endocardial Hey gene transcription and extends Bmp2 expression to the ventricular endocardium. This embryonic Notch1-Bmp2-Snail1 relationship may be relevant in adult valve disease, in which decreased NOTCH signaling causes valve mesenchyme cell formation, fibrosis, and calcification.