Endothelin B receptor (ET_B) is a G-protein-coupled heptahelical receptor sharing the same class as endothelin A receptor (ET_A). Both receptors are widely expressed with interconnected functions, particularly in the cardiovascular system. Both ET_A and ET_B initiate downstream signalling through binding to endothelin. Endothelin are 21 amino-acids peptides categorized into three subclasses: ET-1, ET-2, and ET-3 [1]. ET-3 is responsible for the proliferation of pluripotent neural crest cells (NCCs) through its interaction with ET_B to ensure normal intestinal development whereas ET-1 and ET-2 exert vascular functions through ET_A and ET_B binding.

Homozygous ET_B mutation is known to cause Hirschsprung’s disease (HSCR), a well-described aganglionic intestinal disease affecting 1/5000 births globally but with a incidence up to 1/1370 births regionally [2]. HSCR is generally treated with surgical resections; however, increasing understanding on HSCR and ET_B regulatory signalling suggests such treatments may not be sufficiently comprehensive. Recent studies suggested that multiple organ systems maybe be affected in syndromic HSCR patients [3, 4]; up to 30% of HSCR patients are associated with abnormalities in the central nervous, gastrointestinal, genitourinary, endocrinological, immunological, and cardiovascular systems. Some of the HSCR-associated syndromes with cardiac abnormalities 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) [3, 5‐7]. Furthermore, conotruncal heart malformations such as atrioseptal defect (ASD; 2.2%) and ventricular septal defect (VSD; 1.7%) are also noted in subpopulations of HSCR patients [8].

The pathogenesis of heart defects associated with HSCR and ET_B mutation may partially stem from the migration failure of cardiac neural crest cell (CaNCC), a process dictated predominantly by ET-1/ET_A signalling based on current literature [9]. Animals with ET_A mutation have been known to have high neonatal mortality with severe craniofacial and cardiac malformations [10, 11]. Although ET_B mutation is not known to affect CaNCC directly, it has been reported to alter ET-1/ET_A signalling [12]. Consequently, one can expect subtle cardiac changes to occur in ET_B^−/− mutants.

Vascular dysfunction may also predispose ET_B^−/− mutant to suffer to structural changes during development. ET_B’s cardiovascular effects are bimodal, mediating both vasodilation and vasoconstriction through its binding with ET-1 [13‐15], the principal endothelin isoform in the cardiovascular system. ET-1 is secreted by the vascular endothelial cells and endocardial cells of cardiomyocytes [16]. Several studies have demonstrated that activation of ET-1/ET_B pathway yields endothelial vasodilation via nitric oxide (NO), prostacyclin, and endothelium-relaxing factor (EDRF) thus balancing the vasoconstriction mediated by ET-1/ET_A in vascular smooth muscle cells (VSMC). Consequently, ET_B is likely to possess a beneficial role in myocardial circulation and thus end organ perfusion [17‐19]. By the same token, one would expect HSCR patients with homozygous ET_B^−/− mutation to have impaired cardiovascular development and increased risks for hypertension and coronary artery disease due to the uncontrolled ET-1/ET_A stimulation [20].

Although several microscopic and physiologic studies have been conducted to determine the functions and distribution of ET_B receptor, to the best of our knowledge, no macroscopic analyses have been completed to evaluate its effect on cardiac anatomy [4, 15, 21]. We aim to complement prior studies through quantitative analysis on the cardiac changes of the spotting-lethal (sl/sl) rat, a naturally occurring ET_B^−/− animal model of WS-IV, with the phenotype of HSCR, hearing deficits, and white coat colour [22].

In order to acquire detailed yet structurally preserved anatomical information, we adopted X-ray micro-computed tomography (micro-CT) with modified tissue staining techniques [23]. Micro-CT offers three-dimensional (3D) information with high resolution images comparable to the low powered 2D microscopy. 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:

In this study, we demonstrated neonatal ET_B^−/− HSCR model exhibits significant cardiac structural impairment. This was consistently illustrated through three parameters taken in sl/sl rat: up to 40% volumetric reductions in heart structures, 20% reduction in heart growth rate, and 25% reduction in organ volume/bodyweight indices. The causes for these cardiac growth restrictions likely included the following: global growth impairments due to enteric dysfunction; alterations in CaNCC development; vasodysregulation caused by the absence of ET_B.

Concurrent developmental anomalies occurred in up to 30% of HSCR patients, five to eight percent of whom suffer congenital heart diseases (CHD) [28]. While CHD occurred in only three percent of non-syndromic HSCR infants, the prevalence of CHD in chromosomal HSCR patients is remarkably high, ranging from 20 to 80%, with cardiac septal defects being the most common anomalies [5]. Furthermore, regional paediatric data demonstrated that up to 48% of HSCR patients co-affected with Down’s syndrome (HSCR/DS) also have CHD [29]; this high congruence suggested defects in cardiac and central nervous systems may be closely related in certain HSCR subtypes. Indeed, in addition to the cardiac structural abnormalities reported in this study, we have previously found brain shrinkage (up to 20% reduction) in sl/sl rat despite preservation of normal organ morphology. Our findings suggested HSCR patients, at least in ET_B^−/− variant, are likely to share subtle cardiac and neurological impairments. This also suggested that CHD incidence in HSCR is likely underestimated due to under-reporting of subtle cardiac anomalies.

Homozygous ET_B knock-out mutation (ET_B^−/−) causes WS-IV syndrome with prominent HSCR phenotypes. As previously mentioned, aberrant mutation in ET-3/ET_B signalling causes ENCC migration failure and thus ENS maldevelopment, which impairs the nutritional absorption in affected individuals [30]. Consistently, a 16.53% decrease in bodyweight and a 3.42% decrease in body growth-rate were recorded in sl/sl rat, Fig. 1. These bodily reductions have likely contributed, at least partially, to the proportional shrinkage of the heart. Additionally, this growth retardation will likely worsen with age as the manifestation of enteric failure becomes progressively prominent.

Disruption in CaNCC migration may have contributed to the structural changes of the heart in sl/sl rat. Although limited literature is available to comment on the presence of direct linkage between ET-3/ET_B signalling on CaNCC migration, indirect alteration in ET-1/ET_A signalling through elevated ET-1 levels in ET_B^−/− mutants has been suggested [12]. CaNCC has been documented to colonize cardiac outflow tract (OFT) and pharyngeal arches during embryogenesis. This in turn facilitates the remodelling of pharyngeal arch arteries (PAAs), which gives rise to bilateral carotid arteries, segment of aortic arches, pulmonary artery, and ductus arteriosus [10]. Consequently, removal of CaNCC during development causes inappropriate PAA regression and leads to type b interrupted aortic arch in mouse models [10]. Furthermore, prior studies demonstrated the presence of CaNCC promotes cardiac septation in mouse [31] while its absence leads to ventricular septal defects (VSDs) [11]. Importantly, Clouthier et al. [9] demonstrated mice with defective ET-1/ET_A signalling share features of velocardiofacial syndrome like those of CaNCC migration failure. On the other hand, other than significant size shrinkage, sl/sl rat seemed to exhibit a grossly normal heart; neither large vessel nor cardiac septal anomaly was detected. Although prior lineage-tracing studies did not reveal the participation of CaNCC in the developments of myocardium and epicardium [32], biomarker studies using Plxna2, fate-mapping, and the finding of thin ventricular myocardium as a result of CaNCC gene knock-out studies (e.g. BMPR1A and PAX3) suggested CaNCC contributes to epicardial developments and ventricular myocardium [33‐35]. Concordantly, we found marked atrial and ventricular shrinkage in the heart of sl/sl rat, supporting subtle alterations in CaNCC pathway from ET-1/ET_A hyperstimulation may have partially contributed to the myocardium maldevelopment.

Disruption in ET_B’s vascular control may have also contributed to the detrimental effect of cardiac development in sl/sl rat. ET_B is predominantly expressed in the vascular endothelium where it initiates vasodilation through binding with ET-1, thus triggering decreased clearance of NO, prostacyclin, and EDRF. On the other hand, subtle ET_B presence has also been detected in VSMC, where activation by ET-1 causes vascular constriction; albeit this effect is minor in normal physiological state [14, 17, 18, 36]. Nilsson et al. [37] illustrated ET_B’s functional duality through stronger recordings of vasoconstrictive response in endothelium-denuded porcine coronary artery following stimulation by Sarafotoxin 6c. This showed endothelial ET_B partially regulates baseline vasodilation and basal coronary perfusion, which are vital to the developing heart. Adding insult to injury, the absence of ET_B markedly reduces the clearance of ET-1 and thus increases ET-1 level by up to 6-folds in sl/sl rats [12]. This leads to elevated ET-1/ET_A stimulation and subsequent hyper-vasoconstriction of coronary artery. Consequently, coronary hypoperfusion worsens growth retardation of the heart, as shown by Figs. 3, 4 and 5 [38]. Interestingly, the pattern of stepwise reduction in heart volume, growth rates, organ volume/bodyweight ratios correspond well with decreasing functional ET_B copies, as shown by Additional files 1, 2 and 3: Supplementary Fig. 1–3. This pattern was also consistent with the inverse relationship between myocardial perfusion and the concentration of ET-1 perfusate to coronary artery [38]. Furthermore, Fig. 6 illustrated reduction in ventricular myocardium was slightly more prominent than that of atrial myocardium, which may reflect variance in rat coronary arterial distribution; albeit this regional difference was very small.

Lastly, three-dimensional analyses made on aortic arch demonstrated no conclusive ET_B-dependent relationship, as shown by Figs. 3, 4 and 5. On the other hand, a subtle decrease in aortic luminal diameter was observed in sl/sl rat, as noted by Additional file 4: Supplementary Table 1. This reflected the likely elevated basal vasoconstriction at the time of culling, mediated by elevated ET-1/ET_A signalling. Additionally, this finding was consistent with prior reports that contractile control in large vessels is predominantly ET_A-mediated [13].

Overall, our result showed distinctive difference in cardiac growth between sl/sl and the control groups. The effect of ET_B on cardiogenesis was likely multifactorial rather than a pure manifestation of HSCR’s poor growth [30]. While we cannot be certain on the exact pathogenesis of ET_B dysfunction leading to the cardiac reduction, in conjunction with prior studies, hypoperfusion from vascular dysregulation seems likely. Nevertheless, we acknowledged the possibility of growth impairment from enteric dysfunction and changes in CaNCC colonization to the developing heart, albeit these effects were likely minor if presented. Importantly, our finding provided a clue to the suspected cardiac impairment associated with HSCR, a traditionally thought surgical disease. Indeed, if this quantitative finding is translatable to human, HSCR patients are likely to suffer a significant cardiac structural reduction, ranging from 20 to 40%, and thereby increases risk for development of cardiac failure, at least in the ET_B^−/− subtype. Consequently, a wholistic management may be warranted.

Our findings suggested early screening for cardiovascular risk factors in individuals with HSCR may be beneficial, especially in those with ET_B^−/− subtype. In conjunction to the known vascular dysfunction related to ET_B^−/− mutation, significant structural compromise of the heart in animal model suggested a potential risk for cardiac dysfunction. Thus, a structural review using cardiac magnetic resonance imaging (MRI) followed by a baseline echocardiogram may be beneficial. Additionally, clinical uses of endothelin receptor antagonists should be with increasing caution given the potential adverse effect of ET_B inhibition.

Although we have demonstrated significant structural defects in ET_B^−/− HSCR animals, further studies are required. Firstly, we acknowledge the statistical power can be improved by higher sampling; this can be achieved once a more efficient image segmentation platform becomes available. Secondly, despite image analysis was completed on a pre-set computing algorithm to minimize potential bias, future study should consider adopting additional operators and various software for verification of findings. Furthermore, functional assessments with echocardiograms may be beneficial in establishing the direct correlations between cardiac dysfunction and structural impairments. Lastly, clinical study using cardiac MRI can be beneficial in confirming cardiac defects in HSCR patients and should be considered in future studies.

This study demonstrated a correlation between ET_B function and cardiac size in the sl/sl rat. The exact mechanism of action is unclear at this point but may well be due to an effect on coronary artery tone. Preserved cardiac morphology and aortic arch dimensions suggest impaired CaNCC migration failure may play little role in HSCR. Nevertheless, the finding of significant cardiac size and growth impairment in HSCR model is consistent with an elevated risk of congenital structural heart disease in HSCR patients. It also raises the possibility that ET_B gene may have a role in the development of heart failure, coronary artery disease, and hypertension in the general population.