Hey, There’s a Hole in My Heart
See related article, pages 856–863
Mutations in genes encoding the developmental regulators Notch cause an amalgam of defects such that the many roles of this signaling receptor cannot be teased apart by studying the global loss of Notch alone. Instead more subtle and elegant genetic manipulations are called for to discern the multiple roles of Notch. In the current issue of Circulation Research Fischer et al describe a major step toward the understanding of Notch signaling not through the manipulation of the receptor itself, but by studying the loss of two of its downstream effectors.1 Alone, neither the loss of function of Hey1 nor HeyL appear as necessary contributors to proper heart development. Loss of both Hey1 and HeyL however, leads to atrioventricular (AV) dysplasia and membranous ventricular septal defects during cardiogenesis. These phenotypes mimic common congenital heart defects found in the human population and demonstrate a more accurate mouse model that can be used in studying human disease and genetic anomalies.
Despite medical advances, congenital heart disease among children and infants leading to heart failure is still a common occurrence. In fact congenital heart disease is the most common birth defect among live births with an incidence of roughly 1%.2 In these patients, ventricular septal defects and atrial septal defects are the most frequent forms of congenital heart defects found. Loss of separation between the left and right sides of the heart results in the inefficient recirculation of oxygenated blood. The over-burdened heart becomes enlarged and sets the stage for overall heart failure. In most cases, the underlying molecular basis of these defects is not understood and a means of investigating the molecular etiology of congenital heart disease remains elusive.
Over the past few years, some key discoveries have emerged linking alterations in genes encoding Notch signaling proteins to congenital heart problems. Mutations in the human NOTCH1 gene can cause aortic valve anomalies3 resulting in aortic valve calcification. This study documented the first identification of NOTCH1 germline mutations linked to a human disease. Before this discovery, the human JAGGED1 gene was found by several groups to be linked to Alagille syndrome, a multiorgan disorder involving the liver, eyes, face, skeleton, and notably heart.4 The cardiac defects in Alagille syndrome include ventricular septal defects, however, a majority of patients have right-sided obstructive lesions. A significant amount of morbidity and mortality of Allagille patients are due to cardiovascular anomalies.5 Most recently, the human NOTCH2 gene was also linked to Alagille symdrome.6 Thus, mutations in genes encoding the Notch ligand, Jagged1, or in genes encoding two distinct Notch receptors, Notch1 or Notch2, can lead to abnormal heart development and ultimately heart disease.
Notch functions as a cell surface receptor that becomes a transcriptional regulator when activated by ligand. Indisputably, the best-known targets of Notch signaling are the Hes and Hey family of genes. It is then reasonable to ask the question, is heart development dependent on proper Notch target gene function? Although no Hes/Hey mutations have yet been linked to human cardiovascular disorders, analysis of mouse models has uncovered varied and, in some cases, complementary functions for these genes during early heart development. Furthermore, it is striking that mutations in these murine genes gives rise to congenital heart defects reminiscent of such disorders found in humans.
Fischer et al began their studies of the little known HeyL by examining the loss of gene expression in mice. Similar to Hey1 knockout mice, HeyL−/− mice displayed no obvious pathologies. Owing to the similar sites of expression and the obvious possibility of functional redundancy of these bHLH family members Fischer et al crossed Hey1−/− and HeyL−/− mice to create Hey1/L DKO. A percentage of Hey1/L DKO mice survived until weaning, dependent on genetic background, with the mutations in the C57Bl/6 background more severely affected. By coupling the elegant use of histological analysis with MRI to document heart defects Fischer and colleagues determined the heart defects of Hey1/L DKO embryos manifested after E13.5. The ventricular septums of these embryos appeared unclosed along with the loss of the membranous portion of the septum. Newborn pulmonary valves appeared thickened as well.
Closer examination of Hey2−/− mice revealed ventricular septal defects similar to those found in the Hey1/L DKO, indicative of a shared pathway to proper heart development. Re-evaluation of normal embryonic hearts uncovered the previously overlooked expression of Hey1, Hey2, and HeyL in the AV cushion along with expression of Notch1, Notch2, and Jagged1. Based on target gene expression, the authors suggest that the Notch ligand Jagged1 can signal to Notch1 and Notch2 in the endocardium of the AV canal, a site representing the primary source of mesenchymal cells that form the membranous septum and valves.
The study provides insight into a vexing issue about Notch signaling; that is, how does the coordinated action of several Notch target genes carry out Notch function. There are multiple Hes/Hey family members.7 Although it is clear that Hes/Hey genes are targets of Notch transcriptional regulation, it is not clear how individual Hes/Heys genes function and how those specific functions are coordinated. In terms of successfully building a mammalian heart, we now know that you need at least three Notch target genes, Hey1, Hey2, and HeyL. It will be a challenge to parse out the individual activities of these factors. In humans, these genes represent intriguing candidates as disease-linked genes, possibly increasing our repertoire of mutated genes known to be involved in congenital heart disease.
In the latter half of Fischer et al, the authors suggest that the function of Notch/Hes/Hey in the endocardium is one of promoting epithelial to mesenchymal transition. Explant cultures were undertaken to more accurately observe the epithelial to mesenchymal transition of the endocardial cells of the AV canal. Overall, the study revealed the inability of mesenchymal transformation by Hey1/L, Hey2, and Notch1 mutant cells. Cells from Notch1−/− explants were the most severely affected. Not only was epithelial to mesenchymal transition ability impaired but also Notch1−/− cells failed to migrate of the AV explant. This suggests that Notch1 plays a hierarchical role and that Hey1, Hey2, and HeyL all participate in epithelial to mesenchymal transition to some extent, as schematized in the above Figure. Greater complexity arises if one attempts to place the ligands in this relationship. JAGGED1 mutations in humans do give rise to septal defects but these are less common than right-sided obstructive lesions. This differs from the mouse models analyzing Notch1/Hes/Hey functions. Clearly, the phenotype of individuals with mutations in ligand genes differs from those found in mice with Notch/Hes/Hey gene mutations.
Another recently published article by High et al provides further insight into the source of heart defects in Notch mutant mammals.8 This report demonstrates that Notch plays a role in differentiation of cardiac neural crest precursors into smooth muscle cells. The Notch target genes implicated in this function are Hrt1, Hrt2, Hrt3, also known as Hey1, Hey2, and HeyL, respectively. Thus, the septal defects found in Hey1/L DKO mutant embryos could also arise from improper activity in cardiac neural crest precursors.
A hole in the heart is a serious matter and the common occurrence of congenital heart disease attests to the multiple ways that heart development may go wrong. In closing, the authors of Fischer et al point out that the Hey mutant phenotypes described represent milder phenotypes than that found in Notch1 mutant hearts. This points to the possibility that defects in Notch signaling, because of mutations in Hey genes, may represent a larger percentage of congenital heart diseases than previously appreciated. If so, our understanding of the molecular nature of human congenital heart disease will be significantly enhanced.
Sources of Funding:
Support by National Institutes of Health grants RO1 HL 62454 (J.K.) and T32 DK07328 (S.A.S.).
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
Fischer A, Steidl C, Wagner TU, Lang E, Jakob PM, Friedl P, Knobeloch KP, Gessler M. Combined loss of Hey1 and HeyL causes congenital heart defects because of impaired epithelial to mesenchymal transition. Circ Res. 2007; 100: 856–863.
Yuan ZR, Kohsaka T, Ikegaya T, Suzuki T, Okano S, Abe J, Kobayashi N, Yamada M. Mutational analysis of the Jagged 1 gene in Alagille syndrome families. Human molecular genetics. 1998; 7: 1363–1369.
Kamath BM, Spinner NB, Emerick KM, Chudley AE, Booth C, Piccoli DA, Krantz ID. Vascular anomalies in Alagille syndrome: a significant cause of morbidity and mortality. Circulation. 2004; 109: 1354–1358.