Highlights:
- ETB-/--HSCR was associated with body-growth impairment; neonatal sl/sl rat has 16% less bodyweight than the control group.
- sl/sl rat has grossly normal cardiac morphology.
- Significant reductions were detected in cardiac structures of sl/sl rats: 40% in whole-heart volume, 20% in growth rate, and 25% in whole-heart/bodyweight ratios. Similar trend was seen with LA, LV, RA, and RV.
- No consistent correlation was observed between ETB genotype and aortic arch sizes.
- These findings supported HSCR patients may have various degrees of neonatal cardiac anomalies; wholistic post-operative care should be considered.
- In consideration of the result of this paper, genotyping study of patients with and without heart failure would be an interesting step study to evaluate possible correlation between ETB genotype and risks of hypertension, ischemic heart disease, exercise capacity, and heart failure.
Hirschsprung’s disease (HSCR), also known as “dilatation and hypertrophy of colon,” was first popularized by Dr. Harald Hirschsprung in 1888 (1). HSCR is a pediatric intestinal aganglionic disease affecting 1/5000 births globally but regional incidence may range from 1/1370 to 1/7165 births (2-5). Although the exact mode of HSCR inheritance is yet to be confirmed, the current view supports non-Mendelian pattern with variable penetrance (6). However, this is likely due to multi-genetic involvement with confounding expression pattern. Overall, it has an 4: 1 male predominance (7). With up to 80-90% of cases diagnosed in the neonatal stage, it is commonly known for its clinical manifestations of pseudo-obstruction and associated complications (8). HSCR is commonly treated with resection of variable lengths of hypoganglionic and aganglionic intestinal segments to avoid potential toxic megacolon (7, 9).
The pathogenesis of HSCR involves the migration failure of enteric neural crest cells (ENCC) to the growing gastrointestinal (GI) tract. Consequently, disrupted developments of myenteric and submucosal plexuses result in variable lengths of hypoganglionic and aganglionic colon (10). Similarly, HSCR associated congenital heart defects, particularly conotruncal heart malformations, can also arise from interruption of cardiac neural crest cells (CaNCC) migration (11-13).
During embryogenesis, the neural crest and its derivatives are under strict regulatory control by several genes, which become the potential targets for maldevelopment. In the case of HSCR, common causes include receptor tyrosine kinase (RET), glial cell line derived neurotrophic factor (GDNF), glycosylphosphophatidylinositol-linked receptor (GFRα1), ET-3, and ETB (14-19). Under normal circumstance, GDNF/GFRα1/RET pathway provides the mitogenic drive for GDNF-dependent progenitors, which are responsible for the normal development of all enteric neurons (20, 21). Concomitantly, endothelin-3/endothelin-B receptor signaling (ET-3/ETB) maintains the migration and the pluripotency of ENCC. In the event of ETB mutation, loss of ET-3/ETB stimulation results in premature differentiation of enteric neuron and thereby migration arrest as crest-derived neuronal precursors. Additionally, deficiency of ET-3/ETB retards smooth muscle development and causes intestinal accumulation of laminin-1, which promotes premature-differentiation of enteric neurons through its α1-subunit (22, 23). On the other hand, cardiac outflow tract development is dependent on the colonization by CaNCC, which is controlled by endothelin-1/endothelin-A receptor signaling (ET-1/ETA) (24, 25). While no common regulatory pathways have been known to directly control both CaNCC and ENCC migration during embryogenesis, ETB may exert indirect effect through ET-1/ETA signaling and thereby affecting heart development.
Clinically, this “not-so-conspicuous” relationship between heart malformation and HSCR is supported by a number of well documented syndromes (26). Indeed, recent studies have suggested multiple organ systems are affected in HSCR patients due to the pleomorphic effects of multi-genetic involvement in HSCR etiology (27-29). Up to 30% of HSCR patients are associated with abnormalities in the central nervous, gastrointestinal, genitourinary, endocrinological, immunological, and cardiovascular systems. These abnormalities are typified by some of the serious clinical syndromes, including: Down’s syndrome (up to 2-10% of HSCR cases), Di George syndrome, Haddad syndrome, Mowat-Wilson syndrome, Type IV Waardenburg syndrome (WS-IV), and McKusick-Kauffman syndrome (MKKS). All of which are known to exhibit potential features of cardiac defects associating with HSCR (7, 26, 27, 30, 31). Additionally, conotruncal heart malformations associating with HSCR were also noted, atrioseptal defect (ASD; 2.2%) and ventricular septal defect (VSD; 1.7%) in particular. These reports support HSCR causation genes may have additional impacts on embryological heart development (32-34).
Although it was reported that up to 50% of familial and 30% of sporadic HSCR cases were due to mutation in RET and GDNF pathways whilst only 5% of HSCR were attributed to ET-3/ETB signaling defects (35-37), we suspect ETB’s importance may be underestimated. Indeed, recent studies from Taiwan (38), China (39), and Korea (40) demonstrated various novel mutations including p.P383_L386delinsP, D241D (c723T>C), N426N (c1278T>C), IVS4-14T>C, L227L(c831A>G), promoter-116C>T, 5’UTR-121G>T, IVS4+62C>A, and IVS5+121G>C in the ETB gene, adding additional mutation locus to existing data. Furthermore, Puffenberger et al (1994) also demonstrated significant increased risks for HSCR development from homozygous and heterozygous W276C missense mutations in ETB genes, 74% and 21% respectively. These evidences suggested the possibility of dose-dependent ETB effect and the likelihood of higher-than-quoted ETB mutation prevalence (41).
Among the causes of HSCR, ETB is perhaps the most interesting due to its wide distribution, conflicting evidence of functions, and the strong vasoregulatory importance of endothelin-systems. ETB is a G-protein-coupled heptahelical receptor sharing the same class as ETA (42). It is expressed in the central nervous system (CNS: medulla oblongata, cerebrum, hippocampus, cerebellum, striatum), gastrointestinal (GI; enteric nervous systems), sensory organs (retina and stria vascularis), and cardiovascular systems (CAS: endocardium and coronary arterial endothelium) (28).
Both ETA and ETB initiate downstream signaling through respective binding with endothelin of different affinity.
Endothelin (ET) was first discovered in 1988 (43). In mammals, ET is first generated in the forms of preproendothelin followed by furin (prohormone convertase)-mediated cleavage to form inactive big-endothelin (44). Subsequently, big-endothelin is metabolized by endothelin converting enzymes-1 or -2 (ECE-1 or -2) to yield 21 amino-acids peptides of 3 classes: ET-1, ET-2, and ET-3 (45-47). ET-3 is responsible for the proliferation of pluripotent neural crest cells (NCCs) through its interaction with ETB to ensure normal intestinal development. ET-1 and ET-2 exert their function mainly in cardiovascular system.
Both ETA and ETB have high affinity to ET-1 for vascular control (48); however, little is known about the structural impact from these interactions. Although subjects with ETB mutation are compatible with life (46), loss of ETA function results in severe craniofacial and cardiac defects due to migration failure of cephalic neural crest cell (CNCC) and CaNCC; neonatal mortality can therefore be high (25).
ETB’s cardiovascular effects are two-folds, mediating both vasodilation and vasoconstriction through the binding with ET-1 (49-52), the principal isoform in the cardiovascular system. It is secreted by the vascular endothelial cells and endocardial cells of cardiomyocytes (53, 54). Several studies have demonstrated that activation of ET-1/ETB pathway yields vasodilation via nitric oxide (NO), prostacyclin, and endothelium-relaxing factor (EDRF) thus balancing the vasoconstriction mediated by ET-1/ETA in vascular smooth muscle cells (VSMC). This suggested ETB may have a beneficial role in myocardial circulation (55-57). Indeed, additional support was shown by the increased vasoconstriction observed in endothelium-denuded coronary artery (58). By the same token, one would expect HSCR patients with homozygous ETB -/- mutation to have impaired cardiovascular development and subsequently higher risks for hypertension, coronary artery disease, and congestive heart failure (59, 60).
Although a number of microscopic and physiologic studies have been conducted to determine the functions of ETB receptor, to the best of our knowledge, no macroscopic analyses have been completed on the effect of the ETB gene on cardiac anatomy (28, 52, 61, 62). We aim to complement this knowledge by quantitatively analyzing the cardiac anatomy of the spotting-lethal (sl/sl) rat, a naturally occurring ETB-/- animal model of WS-IV, with the appearance of HSCR, hearing deficits, and white coat color (63). Based on our segregation analysis, sl/sl rat follows autosomal recessive inheritance (p-value = 0.001) with high genetic penetrance, up to 95% of sl/sl rats exhibited HSCR. Conversely, incidence of rare mutant phenotype was seen in less than 3% of the control group, which consisted of the wild-type and heterozygotes. Consequently, statistical comparison in this study was made between sl/sl and control groups.
To achieve detailed yet structurally preserved anatomical information, we adopted X-ray micro-computed tomography (micro-CT) with modified tissue-staining techniques (64). Micro-CT offers three-dimensional (3D) information with high-resolution images comparable to the low powered 2D microscopy, allowing detailed quantitative analysis. In addition, improvement on imaging analysis software in recent years have rendered detection of subtle volumetric and dimensional changes in cardiac system possible.
In this study, we hypothesize the following:
- ETB-/- HSCR model, sl/sl rat, exhibits minor body-growth impairment in early age.
- Gross cardiac morphology may be preserved in sl/sl rat from intact ET-1/ETA
- However, loss of functional ETB gene may be associated with reduced heart size, growth rate, and heart-volume/bodyweight ratio.
- Cardiac growth may be ETB-dose-dependent.
- Absence of functional ETB has little effect on aortic arch growth.