Intercellular Signaling in Cardiac Development and Disease: The NOTCH pathway
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.
KeywordsVentricles Trabeculation Compaction Cardiomyopathy LVNC NOTCH
We will describe in some detail the process of ventricular chamber development because it is the focus of this review. At E9.5, the process of trabeculation begins in the primitive ventricles. Trabeculae are highly organized sheets of cardiomyocytes that protrude toward the light of the ventricles . Trabeculae are the first structural landmark of the developing ventricles . They increase the ventricular surface and allow the myocardium to grow in the absence of coronary vessels that will be formed later. Trabeculae are important for contractibility, formation of the conduction system, and blood flow direction in the ventricle. Although trabeculae start to form first in the left ventricle and later in the right ventricle, there are no differences between these structures in the two ventricles. Formed trabeculae have no free ends and they make the ventricle appear like a sponge  (Fig. 12.1b). From E10.5 onward, the ventricular myocardium has two well-defined regions, morphologically and molecularly, the outer compact myocardium and the inner trabecular layer. The compact myocardium is less differentiated and shows a higher proliferation rate than the trabecular myocardium . Differentiated cardiomyocytes will give rise to the working myocardium and the conduction system . After completion of ventricular septation around E14.5, further growth and maturation of the ventricular chambers require a complete trabecular remodeling and reorganization into an apico-basal orientation that determines the shape of the ventricles (Fig. 12.1c). Trabeculae become compacted and as a result there is a significant change in the ratio of compact vs. trabecular myocardium in the ventricle. Trabeculae compaction is accompanied by the hypoxia-dependent invasion of the coronary vasculature from the epicardium into the compact layer of the myocardium . As they compact, the intertrabecular recesses are transformed into capillaries of the rising coronary plexus. This is followed by a reorganization of the muscle fibers of the heart that organize in a three-layered spiral around the heart reflecting the twisting pattern of heart contraction . Trabeculae stop proliferating while the proliferation of the compact zone sustains the growth of the chamber and the contribution of the compact layer to the heart becomes more significant than the one of the luminal trabeculations that will eventually disappear. How exactly this happens and what molecular mechanisms drive this process is unknown.
12.2 Left Ventricular Non-compaction (LVNC)
LVNC is a cardiomyopathy of poorly understood etiology that is characterized by prominent and excessive trabeculations with deep recesses in the ventricular wall . LVNC was first diagnosed in 1984, in a 33-year-old woman assessed by echocardiography . Since then, the heterogeneity of its symptoms might have left many LVNC cases undiagnosed , giving a lower estimation of its real impact as a cardiomyopathy. The improvement in imaging technologies and the advent of CMRI allow now a better diagnosis of this disease, and 10 years after the first description, LVNC was included in the World Health Organization (WHO) catalogue of cardiomyopathies . In 2006, the American Heart Association included LVNC to its list of genetic cardiomyopathies caused by an arrest of the normal compaction process of the developing myocardium . LVNC can be almost asymptomatic or manifest as depressed systolic function , accompanied by systemic embolism, malignant arrhythmias, heart failure, and sudden death , although its clinical manifestation and the age of the affected individual at symptom onset are variable. LVNC was shown to be caused by mutations in genes encoding mainly structural proteins of the cardiac muscle, cytoskeleton, nuclear membrane, and chaperone proteins. In general, familial forms of LVNC are transmitted as an autosomal-dominant trait . The underlying molecular mechanisms that produce the structural abnormalities leading to LVNC are still unknown .
12.3 The NOTCH Signaling Pathway
12.4 NOTCH in Ventricular Chamber Development
To precisely determine the contribution of Notch ligands to ventricular chamber development, we bred mice bearing a conditional allele of Mib1 with myocardium-specific Cre driver line cTnT-Cre . Histological examination at E16.5 revealed that Mib1 flox ;cTnT-Cre mutants had a dilated heart with a thin compact myocardium and large, non-compacted trabeculae, protruding toward the ventricular lumen. Morphological and functional analysis using echocardiography and cardiac magnetic resonance imaging (CMRI) showed prominent trabeculations, deep intertrabecular recesses, and a significantly reduced ejection fraction in mutant mice . The hearts of these mice had a ratio of non-compacted to compacted myocardium (non-compaction index, NC) of 2.0, a feature diagnostic of LVNC in humans [11, 17]. These features are all strongly reminiscent of LVNC, establishing Mib1 flox ;cTnT-Cre mice as the first animal model of LVNC . Analysis of chamber markers in E15.5 Mib1 flox ;cTnT-Cre mice revealed expansion of various compact myocardium markers (Hey2, Tbx20 and n-myc) to the trabeculae and reduced expression of the trabecular markers Anf, Bmp10, and Cx40. These markers were normally expressed at earlier stages, suggesting that maintenance of trabecular maturation and patterning is impaired in Mib1 flox ;cTnT-Cre mutants. Likewise, lost or reduced Hey1, Hey3, and EphrinB2 expression in the vessels of the compact myocardium suggested that coronary artery development was defective. Proliferation analysis revealed increased proliferation of trabecular cardiomyocytes in the hearts of E15.5 Mib1 flox ;cTnT-Cre embryos, suggesting this as the cause of the enlarged, non-compacted trabeculae in these mutants. RNA sequencing analysis of E14.5 Mib1 flox ;cTnT-Cre mutant ventricles identified altered expression of 315 genes (132 upregulated and 183 downregulated). The expression of genes involved in the differentiation of cardiac endothelium/endocardium and cardiomyocytes and coronary vasculogenesis was altered. RNA-Seq data also confirmed the in situ hybridization analysis and demonstrated that Mib1 inactivation in the myocardium disrupts the differentiation and maturation of cardiac endothelial cells and cardiomyocytes. This may impact in turn the process of ventricular maturation and compaction. Genetic ablation of Mib1 in the myocardium thus leads to LVNC cardiomyopathy in mice by arresting ventricular trabeculae maturation and compaction and increasing cardiomyocyte proliferation during fetal development .
We next examined whether mutations in the human MIB1 homologue were associated with clinical LVNC and identified two mutations in a cohort of 100 Southern European LVNC cases; one was a heterozygous G to T transversion of nucleotide 2827 in exon 20 (causing a change in amino acid 943 from valine to phenylalanine, the p.Val943Phe mutation). Val943 is located within a region that mediates protein-protein interactions and constitutes the site of MIB1 ubiquitin ligase activity. The second mutation was a heterozygous C to T transition of nucleotide 1587 in MIB1 exon 11 (causing a premature stop codon instead of arginine at position 530 in the MIB1 ankyrin repeats region, the p.Arg530X mutation). These two mutations were tracked back through three and two generations of LVNC-affected individuals, revealing co-segregation with LVNC, confirming the hereditary nature of the disease .
In silico modeling suggested that MIB1 is a homodimer where the p.Val943Phe mutant will alter the alignment of the ring finger domains in heterodimers formed by wild-type and mutant p.Val943Phe MIB1, as well as the angle of interaction with JAG1 of the p.Val943Phe wild-type dimers. This possibility was supported by co-immunoprecipitation experiments with tagged wild-type MIB1 and mutant MIB1 isoforms co-transfected in pair-wise combinations into HEK293 cells. The effect of the MIB1 mutations on Jag1 ubiquitylation was tested also in HEK293 cells. Jag1 ubiquitylation was strongly reduced in cells co-transfected with wild-type MIB1 plus p.Val934Phe or wild-type MIB1 plus the p.Arg530X mutant .
Lastly, to study the effect of these mutant MIB1 variants in vivo, their mRNAs were microinjected into zebra fish embryos expressing GFP in the developing myocardium. Examination of 72 hpf larvae injected with p.Val943Phe or p.Arg530X mRNAs revealed severely disrupted embryonic development, disrupted cardiac looping, and kinked tail, typical of defective Notch activity. To complement these findings, we cocultured Jag1-HEK293 cells expressing MIB1 variants with Notch1-expressing HEK293 cells co-transfected with a Notch luciferase reporter. Jag1-HEK293 cells expressing wild-type MIB1 increased Notch reporter activity, whereas the p.Val934Phe and p.Arg530X mutant forms reduced reporter activity, indicating that both human mutations disrupt Notch signaling .
Accumulated data suggested that both MIB1 mutations result in loss of MIB1 WT function. In the case of p.Arg530X, this would be due to haploinsufficiency caused by insufficient synthesis of WT MIB1 protein; in the case of p.Val934Phe, this would be due to a dominant-negative effect of the mutant protein, titrating down the amount of functional WT MIB1 dimers through heterotypic or homotypic interactions. In both cases, loss of MIB1 function leads to disease inherited in an autosomal-dominant fashion.
12.5 Future Directions and Clinical Implications
The data reviewed here establish the causal role of NOTCH dysregulation in LVNC, a congenital cardiomyopathy that results from a developmental arrest in ventricular maturation and myocardial compaction. These findings will lead to a better diagnosis and risk stratification, allowing timely intervention in LVNC-associated complications. In addition, since other NOTCH pathway elements may be involved in LVNC, they could serve as novel diagnostic or therapeutic disease targets. Further research is required to determine the molecular mechanisms and regulatory interactions underlying NOTCH function in ventricular chamber development using genetically modified mouse models and the full implication of altered NOTCH signaling in human LVNC.
We apologize to those authors whose work was not cited. 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. Luxán 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.
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