Structural heart defects associated with ETB mutation, a cause of Hirschsprung disease

Background HSCR, a colonic neurocristopathy affecting 1/5000 births, is suggested to associate with cardiac septal defects and conotruncal malformations. However, we question subtle cardiac changes maybe more commonly present due to multi-regulations by HSCR candidate genes, in this instance, ETB. To investigate, we compared the cardiac morphology and quantitative measurements of sl/sl rat to those of the control group. Methods Eleven neonatal rats were generated from heterozygote (ETB+/−) crossbreeding. Age and bodyweight were recorded at time of sacrifice. Diffusion-staining protocols with 1.5% iodine solution was completed prior to micro-CT scanning. All rats were scanned using an in vivo micro-CT scanner, Caliper Quantum FX, followed by two quality-control scans using a custom-built ex vivo micro-CT system. All scans were reviewed for gross cardiac dysmorphology. Micro-CT data were segmented semi-automatically post-NLM filtering for: whole-heart, LV, RV, LA, RA, and aortic arch. Measurements were taken with Drishti. Following image analysis, PCR genotyping of rats was performed: five sl/sl rats, three wildtype, and three heterozygotes. Statistical comparisons on organ volume, growth rate, and organ volume/bodyweight ratios were made between sl/sl and the control group. Results Cardiac morphology and constituents were preserved. However, significant volumetric reductions were recorded in sl/sl rats with respect to the control: whole heart (38.70%, p value = 0.02); LV (41.22%, p value = 0.01), RV (46.15%, p value = 0.02), LA (44.93%, p value = 0.06), and RA (39.49%, p value = 0.02). Consistent trend was observed in growth rate (~ 20%) and organ-volume/bodyweight ratios (~ 25%). On the contrary, measurements on aortic arch demonstrated no significant difference among the two groups. Conclusion Despite the presence of normal morphology, significant cardiac growth retardation was detected in sl/sl rat, supporting the likely association of cardiac anomalies with HSCR, at least in ETB−/− subtype. Structural reduction was likely due to a combination of failure to thrive from enteric dysfunction, alterations to CaNCC colonization, and importantly coronary hypoperfusion from elevated ET-1/ETA-mediated hypervasoconstriction. Little correlation was detected between aortic arch development and sl/sl rat, supporting minor ETB role in large vessels. Although further clinical study is warranted, HSCR patients may likely require cardiac assessment in view of potential congenital cardiac defects. Supplementary Information The online version contains supplementary material available at 10.1186/s12872-021-02281-2.


Background
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][6][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][14][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][18][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:

Compliance with ethical practice
All tissues and animals used in this study were handled with care and strict compliance to ACT Health Human Research Ethics Committee (ACTH-HREC) and Australian National University Animal Experimentation Ethics Committee (ANU-AEEC), ethic approval project number A2011/67.

Rat culling and method of euthanasia
All rat specimens used in this study were generated from crossbreeding between the heterozygous (ET B +/− ) parents. This breeding colony was originally derived from a naturally occurring mutation and has been maintained in Australian National University (ANU) over the past 15 years.
Eleven neonatal rats with an average age of 88 h were sacrificed. Individual rat's coat pattern, age, gender, and weight were recorded. These rats were over anaesthetized with 5% isoflurane for 15 min in modified gas chamber prior culling. These rats were culled via abdominal aortotomy following a midline laparotomy of 1 cm using scalpel and iris scissor. Five-millimetre tail-tip of each rat was resected and stored for subsequent genotyping.

Tissue preparation and staining protocols
For successful micro-CT scanning, diffusion staining was performed through the following steps. Firstly, midline thoracotomy of one centimetre was performed on rat carcass to facilitate tissue penetration of staining solution into the cardiac tissue. The thoracotomized bodies were immersed in 10% PBS solution for 30 min to wash out residual bodily fluid. This was followed by tissue fixation in 4% formalin solution for 24 h. Next, formalinfixed tissues were then washed out with graded ethanol (EtOH) series: 20%, 50%, 70%, and 90% for 1 day each. Finally, tissue staining was completed in 1.5% iodine in 90% EtOH for a minimum of seven days prior to micro-CT scanning.

Image acquisition by micro-CT scanning
In this study, all rat samples have been scanned using a commercial in vivo micro-CT scanner, Caliper Quantum FX. Additional quality assurance micro-CT scans were acquired using a custom-built ex vivo micro-CT system by ANU Applied Mathematical Department. The maximal resolution achievable by Caliper Quantum FX was 10 µm/voxel with an efficiency of 4.5 min per scan. The average dataset size was 256 MB. The resultant images were stored as DICOM series and visualized using Drishti, an open-source software [24]. The ex vivo micro-CT system built by ANU Applied Mathematical Department required at least fifteen hours of scanning time with additional eight hours of image-processing time via National Computational Infrastructure (NCI) services. The maximal resolution was 1 µm/voxel; magnification factor was limited by the physical size of the sample. The resultant images were stored as netCDF files and visualized with Drishti [24]. Each dataset has an average size of 12-16 GB.
Due to the limited access to the ex vivo micro-CT scanners, all image data acquired by Caliper Quantum FX were filtered with non-local means (NLM) algorithms to improve image quality [25]. Two sets of ex vivo micro-CT data were acquired for quality control to ensure sufficient anatomical details and image clarity in denoised in vivo micro-CT scans available for quantitative analysis.
Although not ideal, we found image quality of post-processed in vivo micro-CT scans satisfactory for the purposes of this study.

Image segmentation and analysis
Acquired micro-CT data were first denoised using NLM algorithm to improve signal-to-noise ratios and therefore image clarity [25]. This code was implemented on an Intel (R) Core ™ i7-4770 K CPU @3.5 GHz system with 32G of RAM and Nvidia GeForce GTX Titan Black Kepler GK110 architecture running Linux.
Following image filtering, micro-CT data were segmented semi-automatically through individual CT slices for selected organs using Drishti segmentation algorithms [24]. To ensure accuracy, follow-up verification on image segmentation was performed one month after the initial processing: minimal variations were detected. The following cardiac organs were selected for quantitative measurements: whole heart, left atrium (LA), left ventricle (LV), right atrium (RA), right ventricle (RV), and ascending aortic arch (AA). This process was repeated for each structure. Segmentation of the whole heart was first completed to determine potential gross cardiac defects in with ET B −/− mutants. The luminal width of AA was measured at the aortic orifice in axial view for standardize comparison. Three-dimensional (3D) volumetric measurements were completed following sub-segmentation of each structure through volume rendering.
To standardize comparison, the following anatomical definitions were adopted. The pulmonary circulatory inflow was defined by the superior and inferior vena cava orifices to right atrium; the outflow was defined by the pulmonary valve. The systemic circulatory inflow was defined by pulmonary vein orifices to the left atrium whereas the outflow was defined by the aortic valve. Both selections of left and right ventricles have included interventricular septal wall for better definition of organ boundary to standardize comparison. Lastly, for the comparison of AA, the boundary of AA was defined as arterial vessel between the aortic valves to the first branching point, brachiocephalic artery.

H&E light microscopy
H&E light microscopy was completed for two of eleven rats following micro-CT scanning to assess cardiac anatomy presented in micro-CT scans. The following steps were performed. The iodine-stained hearts were sectioned longitudinally into tissue blocks of 4 mm in thickness and placed in cassettes. Contrast washout and dehydration were performed in 90% EtOH for 48 h prior to paraffin embedment at 60 °C. These tissue blocks were then sliced into 4 µm thick tissue-sheets with a microtome. Tissue-sheets were then laid in water-bath of 5-6 °C while being positioned onto labelled-glass slides. These slides were dried overnight at 37 °C.
Progressive H&E staining was completed by placing the slides in alum-hematoxylin solutions until the appearance of dark red colour. Washing and 'bluing' with lithium carbonate solution were then performed. Lastly, washing and counter-staining with 0.5% eosin alcoholic solution were completed.
All H&E slides were reviewed with an Olympus IX71 microscope at 4× magnification.

Genotyping
After the completion of quantitative measurements, genotyping was completed as described in the following section. Three homozygous wild-type (ET B ) rats were identified. The average ages of wild-type, heterozygous, and homozygous mutant rats were 90.7 h, 96 h, and 83.2 h respectively.
PCR genotyping was completed through the following protocols. Five-millimetres tail tips of the eleven rats were lysed using Proteinase K in lysis buffer consisting of 100 mM Tris pH 8, 5 mM EDTA, 0.2% SDS, and 200 mM NaCl in distilled water. The DNA was extracted via vortex heating and centrifuging to separate the DNA-containing supernatants from undigested materials. The supernatants were further vortexed and centrifuged to isolate the DNA pellets. The DNA pellets were then washed with 70% EtOH and dried. The DNA was then suspended and quantified using spectrophotometry. Next, PCR was completed with "Master-Mix" reagent, which included: 10*PCR buffer Qiagen-contained MgCl 2 , dNTP (10 mM), Primer PS7 (33.3 μM; 5′-CCA CTA AGA CCT CCT GGA CT-3′), Primer PS 15 (33.3 μM; 5′-TCA CGA CTT AGA AAG CTA CAC T-3′) and DNA polymerase [26]. Afterward, this Master-Mix reagent was pipetted onto PCR plates filled with the eleven rat DNAs followed by PCR in Veriti 96-Well Thermal-Cycler. Finally, electrophoresis of fourteen DNA samples (eleven test-subject DNA and three controls: wild-type, heterozygote, and sl/sl rat) was run for one hour under the voltage setting of 100 V and current setting of 55 mA, with MassRuler (#SM1263, Fermentas) reference by the side. The resultant electrophoresis gel was visualized with Gel Documentation System DOC-Print VX5 (Vilber Lourmat).

Statistical analysis
Based on the segregation analysis of rat colony data showing strong autosomal recessive inheritance (p value = 0.001) and high genetic penetrance (> 95% exhibits HSCR) of sl/sl rats, statistical comparison using the two-tail t-test was made between the sl/sl (ET B ) to determine the effect of ET B on heart growth. The comparison was made in the following parameters: organ size, organ growth rate, and organ volume/bodyweight ratios. Albeit small, the latter two were made to further standardize comparisons by accounting age and body size variations of individual rat. Additionally, these parameters provided estimating metrics for structural changes upon developmental maturation.
The difference (%) between the control and sl/sl groups were calculated for each parameter. Additionally, the proportionality of individual cardiac substituent with respect to the whole heart (organ/heart ratio) were compared to explore potential regional-dependent effect. Lastly, data of respective wild-types (ET B

Reduced bodyweight and body growth rate in sl/sl rat
Based on our measurements of neonatal rats, ET B mutation was associated with minor changes in body size and body growth rate, at least at the age of eighty hours. As shown by Fig. 1a,

sl/sl rat has grossly normal cardiac morphology
Previous studies have suggested disruptions in the endothelin system may lead to CaNCC migration failure and cause cardiac outflow tract defects [27]. However, thorough reviews of the eleven rat micro-CT scans did not reveal marked dysmorphology in cardiac constituents. As exemplified by the sectional micro-CT slices and H&E scan shown in Fig. 2, sl/sl heart contained all the essential components of a normal heart: aorta, aortic semilunar valve, right and left atrium, right and left ventricles, intact interatrial and interventricular septum, and patent pulmonary vessels. Expectedly, cardiac anatomy of wild-type and heterozygous rats shared the same features. This preliminary finding suggested ET B may have little impact on CaNCC migration.

sl/sl rat has significantly smaller heart (mm 3 )
Following the morphological examinations, volumetric measurements were performed for quantitative comparison. Our data demonstrated homozygous ET B mutation was associated with significant volumetric reductions in the heart and constituents.
As demonstrated by Fig. 3, sl/sl rat has a significantly smaller heart than the control group, up to 38.7% Although not reported by current literature, cardiac size may be dose-dependent to ET B gene copies. As shown by Additional file 1: Supplementary Fig. 1, wild-type rat has the largest heart among the three genotypes; this was followed by heterozygote in the middle and sl/sl rat in the last. Concordantly, LA, RA, LV and RV measurements of wild-types were 42.76-48.09% larger than those of sl/sl rats, whereas those of heterozygotes were only 34.71-44.71% larger.
Overall, we demonstrated neonatal sl/sl rats having approximately 40% smaller hearts with respect to the control group. These differences may widen with age until rats reach maturity. ET B mutation has a negative impact on cardiac growth rate. As shown by Fig. 4, the growth rate of whole heart in sl/sl rat was 23.70% lower than that of control group, p value = 0.05. Significant reductions were also observed in the growth rates of LV (26.39%, p value = 0.02) and RV (31.25%, p value = 0.03). Although not reaching the statistical power, lower growth rates of LA (27.36%, p value = 0.18), RA (23.42%, p-value = 0.10) and AA (9.28%, p value = 0.51) were also detected in the sl/sl rats.
Further analysis showed stepwise decreases in organ growth rates with respect to functional copies of ET B gene. As shown by Additional file 2: Supplementary  Fig. 2, LA, RA, LV, and RV growth rates of heterozygous rats were 17.40-26.60% higher than those of sl/sl rats, whereas those of wild-types were 29.43-35.91% higher. On the other hand, analyses on AA growth rates demonstrated no consistent correlations with ET B dose.
Overall, an approximately 25% decrease in the growth rates of all heart constituents could be appreciated in neonatal sl/sl rats. This growth retardation may persist until age of maturation. Interestingly, this reduction was disproportionally larger than the 3.53% reduction in bodyweight growth rate, suggesting an intrinsic effect of ET B to the developing heart.

sl/sl rat has lower heart organ volume/bodyweight indices
Cardiac organ volume/bodyweight ratios demonstrated that effect of ET B mutation on heart structure was disproportionally larger than its effect on body size. Figure 5 showed sl/sl rat has approximately 20% smaller Although not achieving statistical significance, LA volume/bodyweight ratio of sl/sl rat also has a reduction of 25.75%, p value = 0.13. Overall, homozygous ET B mutation was associated with a disproportionally larger reduction in cardiac sizes with respect to changes in global body size. This was further supported by a stepwise decreasing pattern with reducing copies of functional ET B gene, as illustrated by Additional file 3: Supplementary Fig. 3. On the other hand, little change was detected in AA measurements, with sl/sl rat having 5.17% larger AA than that of the control group, p value = 0.70; this observation was consistent with ET B 's minor regulatory role on large vessel.

Uniform structural changes associated with ET B mutation
Subtle variations in the structural changes of different heart components were detected in sl/sl rat. The differences in cardiac constituent/whole heart ratios were relatively uniform between sl/sl rat and the control group, as showed by Fig. 6, suggesting the effect of ET B mutation on the heart was likely global. Overall, these differences between the two groups were insignificant albeit a 5.81% reduction was detected in the RV/whole heart ratio of sl/sl rat.

Discussion
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 . This reflects ET B effect was overall uniform across all heart structures, albeit minor regional-dependent impact might be present in RV 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 knockout studies (e.g. BMPR1A and PAX3) suggested CaNCC contributes to epicardial developments and ventricular myocardium [33][34][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 hypervasoconstriction 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.

Limitations
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.

Conclusion
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.
Additional file 1: Figure 1. Stepwise structural shrinkages were associated with decreasing functional ET B . Stepwise reduction in cardiac size was associated with decreasing ET B copies, with wild-type having largest heart and constituents, followed by heterozygotes in the middle, and sl/sl having the smallest structures: whole heart (55.83 mm 3  Stepwise growth rate reduction was associated with decreasing functional ET B . Stepwise decrease in cardiac growth rates corresponded to reducing ET B copies. As shown, wild-type has highest growth rates, followed by heterozygotes in the middle, and lowest in sl/sl rat: whole heart (0.61 mm 3 /Hr; 0.57 mm 3 /Hr; 0.48 mm 3 /Hr), LV(0.33 mm 3 / Hr; 0.29 mm 3 /Hr; 0.24 mm 3 /Hr), RV (0.24 mm 3 /Hr; 0.23 mm 3 /Hr; 0.18 mm 3 / Hr), LA (0.027 mm 3 /Hr; 0.024 mm 3 /Hr; 0.020 mm 3 /Hr), and RA (0.049 mm 3 / Hr; 0.044 mm 3 /Hr; 0.038 mm 3 /Hr). Wild-type and heterozygotes have relatively small growth rate variations when comparing their respective differences to sl/sl rat. No consistent correlations with genotype can be deduced from aortic arch measurements: wild-type (0.015 mm 3 /Hr), heterozygotes (0.018 mm 3 /Hr), and sl/sl (0.015 mm 3  Additional file 3: Figure 3. stepwise reduction in organ/bodyweight ratio associated with decreasing copies of functional ET B . Positive correlations between cardiac organ/bodyweight indices and ET B gene copy was demonstrated. Wild-type has the highest ratio, followed by heterozygotes in the middle, and sl/sl having the lowest: whole heart (4.31 mm 3 /g; 4.00 mm 3 /g; 3.46 mm 3 /g), LV(2.30 mm 3 /g; 2.02 mm 3 /g; 1.77 mm 3 /g), RV (1.71 mm 3 /g; 1.59 mm 3 /g; 1.31 mm 3 /g), LA (0.19 mm 3 /g; 0.17 mm 3 /g; 0.14 mm 3 /g), and RA (0.34 mm 3 /g; 0.31 mm 3 /g; 0.27 mm 3 /g). Minor difference was presented between wild-type and heterozygote when comparing to the reductions observed in sl/sl rats. In conjunction with Figure 1, this supported ETB mutation has intrinsic effect on cardiac development. No consistent trend associating with genotype can be deduced from aortic arch measurements: wild-type (0.11 mm 3 /g), heterozygotes (0.13 mm 3 /g), and sl/sl (0.  Table 1: sl/sl rat has reduced luminal width of aortic arch when comparing to the control groups. Two-dimensional measurement at the entry of aortic valve showed sl/sl rat having smaller intravascular width comparing to the control group. This trend persisted when respective age and bodyweight were standardized, as shown by the comparison of width growth-rate and width/bodyweight ratio, suggesting possible vasoconstriction.