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Abstract Background The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is uncertain but mitochondrial dysfunction may play an important role. Ketones promote mitochondrial energy production and membrane stabilization. Results SOD1-G93A transgenic ALS mice were fed a ketogenic diet (KD) based on known formulations for humans. Motor performance, longevity, and motor neuron counts were measured in treated and disease controls. Because mitochondrial dysfunction plays a central role in neuronal cell death in ALS, we also studied the effect that the principal ketone body, D-β-3 hydroxybutyrate (DBH), has on mitochondrial ATP generation and neuroprotection. Blood ketones were > 3.5 times higher in KD fed animals compared to controls. KD fed mice lost 50% of baseline motor performance 25 days later than disease controls. KD animals weighed 4.6 g more than disease control animals at study endpoint; the interaction between diet and change in weight was significant (p = 0.047). In spinal cord sections obtained at the study endpoint, there were more motor neurons in KD fed animals (p = 0.030). DBH prevented rotenone mediated inhibition of mitochondrial complex I but not malonate inhibition of complex II. Rotenone neurotoxicity in SMI-32 immunopositive motor neurons was also inhibited by DBH. Conclusion This is the first study showing that diet, specifically a KD, alters the progression of the clinical and biological manifestations of the G93A SOD1 transgenic mouse model of ALS. These effects may be due to the ability of ketone bodies to promote ATP synthesis and bypass inhibition of complex I in the mitochondrial respiratory chain.

The cardiac outflow tract (OFT) transiently links the ventricles to the aortic sac and forms the arterial valves. Abnormalities in these valves, such as bicuspid aortic valve (BAV), are common congenital anomalies. GATA6-inactivating variants cause cardiac OFT defects and BAV, but their mechanisms are unclear. We generated Gata6STOP/+ mice using CRISPR-Cas9, which show highly penetrant BAV (70%) and membranous ventricular septal defects (43%). These mice exhibited decreased proliferation and increased ISL1-positive progenitor cells in the OFT, indicating abnormal cardiovascular differentiation. Gata6 deletion with the Mef2cCre driver line recapitulated Gata6STOP/+ phenotypes, indicating a cell-autonomous role for Gata6 in the second heart field. Gata6STOP/+ mice showed reduced OFT length and caliber, associated with deficient cardiac neural crest cell contribution, which may cause valvulo-septal defects. RNA-sequencing analysis showed depletion in pathways related to cell proliferation and migration, highlighting Cxcr7 (also known as Ackr3) as a candidate gene. Reduced mesenchymal cell migration and invasion were observed in Gata6STOP/+ OFT tissue. CXCR7 agonists reduced mesenchymal cell migration and increased invasion in wild-type but not in Gata6STOP/+ explants, indicating the GATA6-dependent role of CXCR7 in OFT development and its potential link to BAV., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Genome-wide association studies and experimental mouse models implicate the MIB1 and GATA6 genes in congenital heart disease (CHD). Their close physical proximity and conserved synteny suggest that these two genes might be involved in analogous cardiac developmental processes. Heterozygous Gata6 loss-of-function mutations alone or humanized Mib1 mutations in a NOTCH1-sensitized genetic background cause bicuspid aortic valve (BAV) and a membranous ventricular septal defect (VSD), consistent with MIB1 and NOTCH1 functioning in the same pathway. To determine if MIB1-NOTCH and GATA6 interact in valvular and septal development, we generated compound heterozygote mice carrying different Mib1 missense ( Mib1 K735R and Mib1 V943F ) or nonsense ( Mib1 R530X ) mutations with the Gata6 STOP/+ heterozygous null mutation. Combining Mib1 R530X/+ or Mib1 K735R/+ with Gata6 STOP/+ does not affect Gata6 STOP/+ single mutant phenotypes. In contrast, combining Mib1 V943F/+ with Gata6 STOP/+ decreases the incidence of BAV and VSD by 50%, suggesting a suppressive effect of Mib1 V943F/+ on Gata6 STOP/+ . Transcriptomic and functional analyses revealed that while the EMT pathway term is depleted in the Gata6 STOP/+ mutant, introducing the Mib1 V943F variant robustly enriches this term, consistent with the Mib1 V943F/+ phenotypic suppression of Gata6 STOP/+ . Interestingly, combined Notch1 and Gata6 insufficiency led to a nearly fully penetrant VSD but did not affect the BAV phenotype, underscoring the complex functional relationship between MIB1, NOTCH, and GATA6 in valvular and septal development.

Background: Cardiac ventricles provide the contractile force of the beating heart throughout life. How the primitive endocardium-layered myocardial projections called trabeculae form and mature into the adult ventricles is of great interest for biology and regenerative medicine. Trabeculation is dependent on the signaling protein Nrg1 (neuregulin-1). However, the mechanism of action of Nrg1 and its role in ventricular wall maturation are poorly understood., Methods: We investigated the functions and downstream mechanisms of Nrg1 signaling during ventricular chamber development using confocal imaging, transcriptomics, and biochemical approaches in mice with cardiac-specific inactivation or overexpression of Nrg1., Results: Analysis of cardiac-specific Nrg1 mutant mice showed that the transcriptional program underlying cardiomyocyte-oriented cell division and trabeculae formation depends on endocardial Nrg1 to myocardial ErbB2 (erb-b2 receptor tyrosine kinase 2) signaling and phospho-Erk (phosphorylated extracellular signal-regulated kinase; pErk) activation. Early endothelial loss of Nrg1 and reduced pErk activation diminished cardiomyocyte Pard3 and Crumbs2 (Crumbs Cell Polarity Complex Component 2) protein and altered cytoskeletal gene expression and organization. These alterations are associated with abnormal gene expression related to mitotic spindle organization and a shift in cardiomyocyte division orientation. Nrg1 is crucial for trabecular growth and ventricular wall thickening by regulating an epithelial-to-mesenchymal transition-like process in cardiomyocytes involving migration, adhesion, cytoskeletal actin turnover, and timely progression through the cell cycle G2/M phase. Ectopic cardiac Nrg1 overexpression and high pErk signaling caused S-phase arrest, sustained high epithelial-to-mesenchymal transition-like gene expression, and prolonged trabeculation, blocking compact myocardium maturation. Myocardial trabecular patterning alterations resulting from above- or below-normal Nrg1-dependent pErk activation were concomitant with sarcomere actin cytoskeleton disorganization. The Nrg1 loss- and gain-of-function transcriptomes were enriched for Yap1 (yes-associated protein-1) gene signatures, identifying Yap1 as a potential downstream effector. Furthermore, biochemical and imaging data reveal that Nrg1 influences pErk activation and Yap1 nuclear-cytoplasmic distribution during trabeculation., Conclusions: These data establish the Nrg1-ErbB2/ErbB4-Erk axis as a crucial regulator of cardiomyocyte cell cycle progression and migration during ventricular development., Competing Interests: Disclosures None.

Importance: Nonsyndromic bicuspid aortic valve (nsBAV) is the most common congenital heart valve malformation. BAV has a heritable component, yet only a few causative genes have been identified; understanding BAV genetics is a key point in developing personalized medicine., Objective: To identify a new gene for nsBAV., Design, Setting, and Participants: This was a comprehensive, multicenter, genetic association study based on candidate gene prioritization in a familial cohort followed by rare and common association studies in replication cohorts. Further validation was done using in vivo mice models. Study data were analyzed from October 2019 to October 2022. Three cohorts of patients with BAV were included in the study: (1) the discovery cohort was a large cohort of inherited cases from 29 pedigrees of French and Israeli origin; (2) the replication cohort 1 for rare variants included unrelated sporadic cases from various European ancestries; and (3) replication cohort 2 was a second validation cohort for common variants in unrelated sporadic cases from Europe and the US., Main Outcomes and Measures: To identify a candidate gene for nsBAV through analysis of familial cases exome sequencing and gene prioritization tools. Replication cohort 1 was searched for rare and predicted deleterious variants and genetic association. Replication cohort 2 was used to investigate the association of common variants with BAV., Results: A total of 938 patients with BAV were included in this study: 69 (7.4%) in the discovery cohort, 417 (44.5%) in replication cohort 1, and 452 (48.2%) in replication cohort 2. A novel human nsBAV gene, MINDBOMB1 homologue MIB1, was identified. MINDBOMB1 homologue (MIB1) is an E3-ubiquitin ligase essential for NOTCH-signal activation during heart development. In approximately 2% of nsBAV index cases from the discovery and replication 1 cohorts, rare MIB1 variants were detected, predicted to be damaging, and were significantly enriched compared with population-based controls (2% cases vs 0.9% controls; P = .03). In replication cohort 2, MIB1 risk haplotypes significantly associated with nsBAV were identified (permutation test, 1000 repeats; P = .02). Two genetically modified mice models carrying Mib1 variants identified in our cohort showed BAV on a NOTCH1-sensitized genetic background., Conclusions and Relevance: This genetic association study identified the MIB1 gene as associated with nsBAV. This underscores the crucial role of the NOTCH pathway in the pathophysiology of BAV and its potential as a target for future diagnostic and therapeutic intervention.

Background: The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes., Methods: We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3 , APCDD1 , TMX3, CEP192 , and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot., Results: Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3 , APCDD1 , TMX3, CEP192 , and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation., Conclusions: These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.

Mutations in the G protein–coupled receptor GPR126/ADGRG6 cause human diseases, including defective peripheral nervous system (PNS) myelination. To study GPR126 function, we generated new genetic mice and zebrafish models. Murine Gpr126 is expressed in developing heart endocardium, and global Gpr126 inactivation is embryonically lethal, with mutants having thin-walled ventricles but unaffected heart patterning or maturation. Endocardial-specific Gpr126 deletion does not affect heart development or function, and transgenic endocardial GPR126 expression fails to rescue lethality in Gpr126 -null mice. Zebrafish gpr126 mutants display unaffected heart development. Gpr126 is also expressed in placental trophoblast giant cells. Gpr126 -null mice with a heterozygous placenta survive but exhibit GPR126-defective PNS phenotype. In contrast, Gpr126 -null embryos with homozygous mutant placenta die but are rescued by placental GPR126 expression. Gpr126 -deficient placentas display down-regulation of preeclampsia markers Mmp9 , Cts7 , and Cts8 . We propose that the placenta-heart axis accounts for heart abnormalities secondary to placental defects in Gpr126 mutants.

Objective: Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers., Approach and Results: Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development., Conclusions: Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.

Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which ectopic Bmp2 expression is controlled by the Tie2 promoter. Tie2 CRE/+ ;Bmp2 tg/tg mice develop aortic valve dysfunction postnatally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2 CRE/+ ;Bmp2 tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and genetic bone disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2 CRE/+ ;Bmp2 tg/tg transgenic animals is accompanied by increased bone marrow hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type bone marrow hematopoietic stem cells into lethally irradiated Tie2 CRE/+ ;Bmp2 tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic bone marrow into wild-type recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic bone marrow colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell origin. Thus, Tie2 CRE/+ ;Bmp2 tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation., (© 2021. The Author(s).)

Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. Identification of early predictive markers could help optimize patient management. RNA-sequencing was carried out on human fetal aortic valves at gestational weeks 9 , 13 , and 22 and on a case-control study with adult noncalcified and calcified bicuspid and tricuspid aortic valves. In dimension reduction and clustering analyses, diseased valves tended to cluster with fetal valves at week 9 rather than normal adult valves, suggesting that part of the disease program might be due to reiterated developmental processes. The analysis of groups of coregulated genes revealed predominant immune-metabolic signatures, including innate and adaptive immune responses involving lymphocyte T-cell metabolic adaptation. Cytokine and chemokine signaling, cell migration, and proliferation were all increased in CAVD, whereas oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at fetal stages and increased in adult controls, suggesting that these processes intensify throughout life and heighten in disease. Cellular stress response and neurodegeneration gene signatures were aberrantly expressed in CAVD, pointing to a mechanistic link between chronic inflammation and biological aging. Comparison of the valve RNA-sequencing data set with a case-control study of whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a highly predictive gene signature of CAVD and of moderate aortic valve calcification in overtly healthy individuals. These data deepen and broaden our understanding of the molecular basis of CAVD and identify a peripheral blood gene signature for the early detection of aortic valve calcification.

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade., Competing Interests: ST, VO, BP, JG, RP, VB, MG, FS, DM, Jd No competing interests declared, (© 2019, Travisano et al.)

Cardiogenesis is a complex developmental process involving multiple overlapping stages of cell fate specification, proliferation, differentiation, and morphogenesis. Precise spatiotemporal coordination between the different cardiogenic processes is ensured by intercellular signalling crosstalk and tissue-tissue interactions. Notch is an intercellular signalling pathway crucial for cell fate decisions during multicellular organismal development and is aptly positioned to coordinate the complex signalling crosstalk required for progressive cell lineage restriction during cardiogenesis. In this Review, we describe the role of Notch signalling and the crosstalk with other signalling pathways during the differentiation and patterning of the different cardiac tissues and in cardiac valve and ventricular chamber development. We examine how perturbation of Notch signalling activity is linked to congenital heart diseases affecting the neonate and adult, and discuss studies that shed light on the role of Notch signalling in heart regeneration and repair after injury.

Abnormalities of the arterial valve leaflets, predominantly bicuspid aortic valve, are the commonest congenital malformations. Although many studies have investigated the development of the arterial valves, it has been assumed that, as with the atrioventricular valves, endocardial to mesenchymal transition (EndMT) is the predominant mechanism. We show that arterial is distinctly different from atrioventricular valve formation. Whilst the four septal valve leaflets are dominated by NCC and EndMT-derived cells, the intercalated leaflets differentiate directly from Tnnt2-Cre +/Isl1+ progenitors in the outflow wall, via a Notch-Jag dependent mechanism. Further, when this novel group of progenitors are disrupted, development of the intercalated leaflets is disrupted, resulting in leaflet dysplasia and bicuspid valves without raphe, most commonly affecting the aortic valve. This study thus overturns the dogma that heart valves are formed principally by EndMT, identifies a new source of valve interstitial cells, and provides a novel mechanism for causation of bicuspid aortic valves without raphe., Competing Interests: LE, AA, DM, RR, LM, AS, MS, ST, LM, AG, Jd, BC, DH No competing interests declared, (© 2018, Eley et al.)

Aim: To determine the role of NOTCH during the arterial injury response and the subsequent chronic arterial-wall inflammation underlying atherosclerosis., Methods and Results: We have generated a mouse model of endothelial-specific (Cdh5-driven) depletion of the Notch effector recombination signal binding protein for immunoglobulin kappa J region (RBPJ) [(ApoE-/-); homozygous RBPJk conditional mice (RBPJflox/flox); Cadherin 5-CreERT, tamoxifen inducible driver mice (Cdh5-CreERT)]. Endothelial-specific deletion of RBPJ or systemic deletion of Notch1 in athero-susceptible ApoE-/- mice fed a high-cholesterol diet for 6 weeks resulted in reduced atherosclerosis in the aortic arch and sinus. Intravital microscopy revealed decreased leucocyte rolling on the endothelium of ApoE-/-; RBPJflox/flox; Cdh5-CreERT mice, correlating with a lowered content of leucocytes and macrophages in the vascular wall. Transcriptome analysis revealed down-regulation of proinflammatory and endothelial activation pathways in atherosclerotic tissue of RBPJ-mutant mice. During normal Notch activation, Jagged1 signalling up-regulation in endothelial cells promotes nuclear translocation of the Notch1 intracellular domain (N1ICD) and its physical interaction with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). This N1ICD-NF-κB interaction is required for reciprocal transactivation of target genes, including vascular cell adhesion molecule-1., Conclusions: Notch signalling pathway inactivation decreases leucocyte rolling, thereby preventing endothelial dysfunction and vascular inflammation. Attenuation of Notch signalling might provide a treatment strategy for atherosclerosis.

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., (© 2016 American Heart Association, Inc.)

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., (© 2016 American Heart Association, Inc.)

The Notch signalling pathway plays crucial roles in cardiac development and postnatal cardiac homoeostasis. Gain- and loss-of-function approaches indicate that Notch promotes or inhibits cardiogenesis in a stage-dependent manner. However, the molecular mechanisms are poorly defined because many downstream effectors remain to be identified. Genome-scale analyses are shedding light on the genes that are regulated by Notch signalling and the mechanisms underlying this regulation. We review the functional data that implicates Notch in cardiac morphogenetic processes and expression profiling studies that enlighten the regulatory networks behind them. A recurring theme is that Notch cross-talks reiteratively with other key signalling pathways including Wnt and Bmp to coordinate cell and tissue interactions during cardiogenesis.

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.

Objective: Calcific aortic valve disease is similar to atherosclerosis in that both diseases result from chronic inflammation and endothelial dysfunction. Heterozygous NOTCH1 mutations have been associated to calcific aortic disease and a bicuspid aortic valve. We investigated whether mice with genetic inactivation of the Notch signaling pathway are prone to develop valve disease when exposed to a predisposing diet., Methods and Results: Using Doppler echocardiography, histology, immunohistochemistry, quantitative gene expression analysis, and cell culture assays, we examined the effect of a hypercholesterolemic diet supplemented with vitamin D on mice heterozygous for null mutations in the Notch1 receptor or the effector transcription factor gene RBPJk. After 16 weeks on the hyperlipidemic diet, calcific aortic disease was detected in heterozygous RBPJk mice. Analysis of valve leaflets revealed macrophage infiltration, enhanced collagen deposition, proosteogenic protein expression, and calcification. Heterozygous null Notch1 mice displayed milder histopathologic changes and did not develop any significant hemodynamic disturbance. Valvular disease correlated with reduced expression of the Notch target gene Hey1 in valves of RBPJk heterozygous mice fed the hyperlipidemic diet. Consistent with the in vivo data, Notch signaling inhibition in porcine valve interstitial cells led to downregulation of HEY1 transcription, activation of osteogenic markers, and increased calcified nodule formation., Conclusions: We show that Notch signaling disruption via RBPJk heterozygous inactivation results in aortic valve disease. Notch1 heterozygous mice do not show functional impairment, suggesting that additional Notch receptors may be involved in aortic valve homeostasis and disease. Our data establish a genetic mouse model of calcific aortic valve disease and may help to identify a patient population with reduced valvular NOTCH signaling at risk for developing this disease.

The Notch pathway is an intercellular signaling mechanism involved in multiple cell-to-cell communication processes that regulate cell fate specification, differentiation, and tissue patterning during embryogenesis and adulthood. Functional studies in the mouse have shown that a Hey-Bmp2 regulatory circuit restricts Bmp2 expression to presumptive valve myocardium (atrioventricular canal and outflow tract). Likewise, a Notch-Hey-Bmp2 axis represses Bmp2 in the endocardium. During cardiac valve formation, endocardial Notch signaling activates the epithelial-mesenchyme transition (EMT) that will give rise to the cardiac valve primordia. During this process, Notch integrates with myocardially derived signals (Bmp2 or Bmp4) to promote, via Snail1/2 activation a complete, invasive EMT in presumptive valve tissue. In humans, mutations in Notch signaling components are associated with several congenital disorders involving malformed valves, aortic arch, and defective chamber septation. Data suggest that the same embryonic Notch-Hey-Bmp2 regulatory axis is active in the adult valve. This review examines the experimental evidence supporting a role for Notch in heart valve development and homeostasis, and how altered Notch signaling may lead to valve disease in the newborn and adult., (Copyright © 2011 Wiley-Liss, Inc.)

Rationale: The proepicardium is a transient structure comprising epicardial progenitor cells located at the posterior limit of the embryonic cardiac inflow. A network of signals regulates proepicardial cell fate and defines myocardial and nonmyocardial domains at the venous pole of the heart. During cardiac development, epicardial-derived cells also contribute to coronary vessel morphogenesis., Objective: To study Notch function during proepicardium development and coronary vessel formation in the mouse., Methods and Results: Using in situ hybridization, RT-PCR, and immunohistochemistry, we find that Notch pathway elements are differentially activated throughout the proepicardial-epicardial-coronary transition. Analysis of RBPJk-targeted embryos indicates that Notch ablation causes ectopic procardiogenic signaling in the proepicardium that in turn promotes myocardial differentiation in adjacent mesodermal progenitors, resulting in a premature muscularization of the sinus venosus horns. Epicardium-specific Notch1 ablation using a Wt1-Cre driver line disrupts coronary artery differentiation, reduces myocardium wall thickness and myocyte proliferation, and reduces Raldh2 expression. Ectopic Notch1 activation disrupts epicardium development and causes thinning of ventricular walls., Conclusions: Epicardial Notch modulates cell differentiation in the proepicardium and adjacent pericardial mesoderm. Notch1 is later required for arterial endothelium commitment and differentiation and for vessel wall maturation during coronary vessel development and myocardium growth.

The Notch-signaling pathway is involved in multiple processes during vertebrate cardiac development. Cardiomyocyte differentiation, patterning of the different cardiac regions, valve development, ventricular trabeculation, and outflow tract development have all been shown to depend on the activity of specific Notch-signaling elements. From these studies, it becomes obvious that Notch regulates in a cell autonomous or non-cell autonomous manner different signaling pathways, pointing to a role for Notch as a signal coordinator during cardiogenesis. While most of the research has concentrated on Notch signaling in the myocardium, the importance of Notch activity in the cardiac endothelium (endocardium) must not be overlooked. Endocardial Notch activity is crucial for valve and ventricular trabeculae development, two processes that illustrate the role of Notch as a signal coordinator. The importance of Notch signaling in human disease is evident from the discovery that many mutations in components of this pathway segregate in several inherited and acquired disorders. This reflects the fundamental roles that Notch performs during cardiac ontogeny. This review examines the experimental evidence supporting a role for Notch in cardiac development and adult heart homeostasis, and how dysregulated Notch signaling may lead to cardiac disease in the newborn and in the adult., (Copyright 2010 Elsevier Inc. All rights reserved.)

Nicotinamide adenine dinucleotide (NAD)+-dependent sirtuins have been identified to be key regulators in the lifespan extending effects of calorie restriction (CR) in a number of species. In this study we report for the first time that promotion of the NAD+-dependent sirtuin, SIRT1-mediated deacetylase activity, may be a mechanism by which CR influences Alzheimer disease (AD)-type amyloid neuropathology. Most importantly, we report that the predicted attenuation of beta-amyloid content in the brain during CR can be reproduced in mouse neurons in vitro by manipulating cellular SIRT1 expression/activity through mechanisms involving the regulation of the serine/threonine Rho kinase ROCK1, known in part for its role in the inhibition of the non-amyloidogenic alpha-secretase processing of the amyloid precursor protein. Conversely, we found that the expression of constitutively active ROCK1 in vitro cultures significantly prevented SIRT1-mediated response, suggesting that alpha-secretase activity is required for SIRT1-mediated prevention of AD-type amyloid neuropathology. Consistently we found that the expression of exogenous human (h) SIRT1 in the brain of hSIRT1 transgenics also resulted in decreased ROCK1 expression and elevated alpha-secretase activity in vivo. These results demonstrate for the first time a role for SIRT1 activation in the brain as a novel mechanism through which CR may influence AD amyloid neuropathology. The study provides a potentially novel pharmacological strategy for AD prevention and/or treatment.

The human L3MBTL gene is located in 20q12, a region that is commonly deleted in myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). L3MBTL is highly homologous to the D-lethal(3) malignant brain tumor [D-l(3)mbt] gene, which is a putative tumor-suppressor gene (TSG) identified in Drosophila and which is closely related to the Drosophila sex combs on midleg (SCM) protein, a member of the Polycomb group (PcG) family of transcriptional repressors. To examine whether L3MBTL functions as a "classic" TSG in human hematologic malignancies, we screened a panel of 17 myeloid leukemia cell lines and peripheral blood or bone marrow samples from 29 MDS and 13 MPD patients for mutations in the entire L3MBTL coding sequence, including intron/exon splice junctions. No mutations were identified, although two single nucleotide differences were found (in intron 14 and in exon 15), which were interpreted as polymorphic changes. We used real-time RT-PCR to quantify the level of L3MBTL mRNA in various normal myeloid and lymphoid cell populations. L3MBTL is expressed in normal CD34+ bone marrow cells, and we found that the pattern of L3MBTL expression was similar to that of BMI1, a well-studied PcG gene with oncogenic activity, suggesting that L3MBTL and BMI1 may be co-regulated during hematopoiesis. The expression of L3MBTL mRNA in 30 of 35 cell lines and 13 of 15 AML samples was comparable to the level of L3MBTL expression in the normal cell populations. However, five leukemia cell lines showed no L3MBTL expression, and two of the AML samples showed aberrant L3MBTL expression. These data suggest that L3MBTL is not mutated in MDS or MPD. However, given the known dosage effects of PcG proteins in regulating gene expression, reduced or absent L3MBTL expression may be relevant in some cases of myeloid leukemia., (Copyright 2004 Wiley-Liss, Inc.)

We report on the X-ray structure of three 100-amino acid mbt repeats in h-l(3)mbt, a polycomb group protein involved in transcriptional repression, whose gene is located in a region of chromosome 20 associated with hematopoietic malignancies. Interdigitation between the extended arms and cores of the mbt repeats results in a three-leaved propeller-like architecture, containing a central cavity. We have identified one ligand binding pocket per mbt repeat, which accommodates either the morphilino ring of MES or the proline ring of the C-terminal peptide segment, within a cavity lined by aromatic amino acids. Strikingly, phenotypic alterations resulting from point mutations or deletions in the mbt repeats of the related Drosophila SCM protein are clustered in and around the ligand binding pocket.

H-L(3)MBT, the human homolog of the Drosophila lethal(3)malignant brain tumor protein, is a member of the polycomb group (PcG) of proteins, which function as transcriptional regulators in large protein complexes. Homozygous mutations in the l(3)mbt gene cause brain tumors in Drosophila, identifying l(3)mbt as a tumor suppressor gene. The h-l(3)mbt gene maps to chromosome 20q12, within a common deleted region associated with myeloid hematopoietic malignancies. H-L(3)MBT contains three repeats of 100 residues called MBT repeats, whose function is unknown, and a C-terminal alpha-helical structure, the SPM (SCM, PH, MBT domain, which is structurally similar to the SAM (sterile alpha motif) protein-protein interaction domain, found in several ETS transcription factors, including TEL (translocation Ets leukemia). We report that H-L(3)MBT is a transcriptional repressor and that its activity is largely dependent on the presence of a region containing the three MBT repeats. H-L(3)MBT acts as a histone deacetylase-independent transcriptional repressor, based on its lack of sensitivity to trichostatin A. We found that H-L(3)MBT binds in vivo to TEL, and we have mapped the region of interaction to their respective SPM/SAM domains. We show that the ability of TEL to repress TEL-responsive promoters is enhanced by the presence of H-L(3)MBT, an effect dependent on the H-L(3)MBT and the TEL interacting domains. These experiments suggest that histone deacetylase-independent transcriptional repression by TEL depends on the recruitment of PcG proteins. We speculate that the interaction of TEL with H-L(3)MBT can direct a PcG complex to genes repressed by TEL, stabilizing their repressed state.

Erythroid leukemia (ERL or AML-M6) is an uncommon subtype of acute myeloid leukemia, the clinical, morphological, and genetic behavior of which needs further characterization. We analyzed a homogeneous group of 23 de novo AML-M6 patients whose bone marrow cells showed complex karyotypes. We also analyzed eight leukemia cell lines with erythroid phenotype, performing detailed molecular cytogenetic analyses, including spectral karyotyping (SKY) in all samples. The main features are: (1) A majority of patients (56%) had hypodiploidy. Loss of genetic material was the most common genetic change, especially monosomies of chromosome 7 or 18, and deletions of chromosome arm 5q. Taken together, 87% of the cases displayed aberrations involving chromosome 5 or 8. (2) We describe a novel, cryptic, and recurrent translocation, t(11;19)(p11.2;q13.1). Another translocation, t(12;21)(p11.2;q11.2), was found to be recurrent in a patient with ERL and in the K562 cell line. (3) MLL gene rearrangements were detected in 20% of cases (three translocations and three amplifications) and, overall, we defined 52 rearrangements (excluding deletions) with a mean of 2.3 translocations per patient. (4) Of the structural aberrations, 21% involved chromosomes 11 and 19. Most of the rearrangements were unbalanced; only 13 reciprocal translocations were observed. The general picture of chromosomal aberrations in cell lines did not reflect what occurred in patient samples. However, both primary samples and cell lines shared three common breakpoints at 19q13.1, 20q11.2, and 21q11.2. This is the first molecular cytogenetic description of the karyotype abnormalities present in patients with ERL. It should assist in the identification of genes involved in erythroleukemogenesis., (Copyright 2003 Wiley-Liss, Inc.)

The CCAAT/enhancer binding protein alpha (C/EBPalpha) belongs to a family of transcription factors that are involved in the differentiation process of numerous tissues, including the liver and hematopoietic cells. C/EBPalpha(-/-) mice show a block in hematopoietic differentiation, with an accumulation of myeloblasts and an absence of mature granulocytes, whereas expression of C/EBPalpha in leukemia cell lines leads to granulocytic differentiation. Recently, dominant-negative mutations in the C/EBPalpha gene and down-regulation of C/EBPalpha by AML1-ETO, an AML associated fusion protein, have been identified in acute myelogenous leukemia (AML). To better understand the role of C/EBPalpha in the lineage commitment and differentiation of hematopoietic progenitors, we transduced primary human CD34(+) cells with a retroviral construct that expresses the C/EBPalpha cDNA fused in-frame with the estrogen receptor ligand-binding domain. Induction of C/EBPalpha function in primary human CD34(+) cells, by the addition of beta-estradiol, leads to granulocytic differentiation and inhibits erythrocyte differentiation. Using Affymetrix (Santa Clara, CA) oligonucleotide arrays we have identified C/EBPalpha target genes in primary human hematopoietic cells, including granulocyte-specific genes that are involved in hematopoietic differentiation and inhibitor of differentiation 1 (Id1), a transcriptional repressor known to interfere with erythrocyte differentiation. Given the known differences in murine and human promoter regulatory sequences, this inducible system allows the identification of transcription factor target genes in a physiologic, human hematopoietic progenitor cell background.