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

Abstract

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, ET_B. 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 (ET_B^+/-) 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 ET_B^-/- 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/ET_A-mediated hypervasoconstriction. Little correlation was detected between aortic arch development and sl/sl rat, supporting minor ET_B role in large vessels. Although further clinical study is warranted, HSCR patients may likely require cardiac assessment in view of potential congenital cardiac defects.

Keywords: Endothelin-B mutation; Heart defects; Hirschsprung disease; Spotting-lethal rat.

Fig. 1

Decreases in bodyweight (A) and body-growth rates (B) in sl/sl rat. A showed sl/sl (ET_B^−/−) rats having statistically significant smaller bodyweight than the control group (ET_B^+/+ and ET_B^+/−), 11.40 g versus 13.26 g, p value = 0.03. However, when corrected with respective age (Hr), smaller difference was observed between the two groups, 0.137 g/h versus 0.142 g/h, p value = 0.58. ET_B^−/− induced global growth impairment was likely small in the early stage of development. *: statistically significant, p value ≤ 0.05

Fig. 2

Grossly normal cardiac morphology and constituents in sl/sl rat. A (4× H&E microscopy), B and C (ex vivo micro-CT, 10.7 µm/voxel) were cardiac illustrations sl/sl rats in coronal views. No detectable gross abnormality was identified. Both image types showed patent cardiac outflow tract with fully formed atria and ventricles. Septal defects were not detected. Major structures were as labelled. Aor = Aorta; RAu = Right Auricle; RA = Right Atrium; RV = Right Ventricle; RVW = Right Ventricular Wall; IVS = Interventricular Septum; LA = Left Atrium; LV = Left Ventricle; LVW = Left Ventricular Wall; Pm = Papillary Muscle; Pul V = Pulmonary Vessel; Asv = Aortic semilunar valve

Fig. 3

sl/sl rat has significantly smaller cardiac structures. sl/sl rat has a smaller heart when comparing to the control animals, 39.81mm³ versus 55.22mm³, p value = 0.02. Analyses on heart constituents showed similar trend: LV (20.30mm³ vs. 28.67mm³, p value = 0.01), RV (15.04mm³ vs. 21.98mm³, p value = 0.02), LA (1.66mm³ vs. 2.40mm³, p value = 0.06), and RA (3.11mm³ vs. 4.33mm³, p value = 0.02). Minor volumetric difference in aortic arch was also detected between sl/sl and the control group: 1.29 mm³ versus 1.56mm³, p value = 0.25. RA = Right Atrium; RV = Right Ventricle; LA = Left Atrium; LV = Left Ventricle. *: statistically significant, p value ≤ 0.05

Fig. 4

sl/sl rat has lower cardiac growth rates than the control group. Markedly lower cardiac growth rate was detected in sl/sl rat with respect to the control animal, 0.48 mm³/h versus 0.59 mm³/h, p value = 0.05. Similar difference was observed between the two groups in LV (0.24mm³/h vs. 0.31mm³/h, p value = 0.02) and RV (0.18mm³/h vs. 0.23mm³/h, p value = 0.03). Albeit not reaching statistical power, decreasing trend was also seen in LA (0.020mm³/h vs. 0.026mm³/h, p value = 0.18) and RA (0.038mm³/h vs. 0.046mm³/h, p value = 0.10). Aortic arch measurement showed little difference between the two groups: 0.015mm³/h versus 0.017mm³/h, p value = 0.51. RA = Right Atrium; RV = Right Ventricle; LA = Left Atrium; LV = Left Ventricle. *: statistically significant, p value ≤ 0.05

Fig. 5

sl/sl rat has disproportionally larger reductions in its heart size with respect to its bodyweight. Markedly smaller cardiac-organ/bodyweight ratios were observed in sl/sl rat when comparing to those of control group: whole heart (3.46 mL/g vs. 4.15 mL/g, p value = 0.04), LV (1.77 mL/g vs. 2.16 mL/g, p value = 0.03), RV (1.31 mL/g vs. 1.65 mL/g, p value = 0.04), LA (0.14 mL/g vs. 0.18 mL/g, p value = 0.13), and RA (0.27 mL/g vs. 0.33 mL/g, p value = 0.049). This trend reflected disproportionally larger structural impairment in sl/sl heart with respect to global growth restriction, supporting an intrinsic effect of ET_B on heart growth. On the contrary, measurement on aortic arch showed little difference between the two groups: 0.112 mL/g versus 0.118 mL/g, p value = 0.70. RA = Right Atrium; RV = Right Ventricle; LA = Left Atrium; LV = Left Ventricle. *: statistically significant in comparison to ET_B^−/− group, p ≤ 0.05

Fig. 6

Cardiac shrinkage in sl/sl rat was relatively uniform across four major constituents. No significant difference was detected in cardiac constituent/whole heart ratios between sl/sl and the control group. Both shared similar organ/whole heart ratios: LV (0.51 vs. 0.52, p value = 0.72), RV (0.38 vs. 0.40, p value = 0.50), LA (0.042 vs. 0.043, p value = 0.95), and RA (0.079 vs. 0.079, p value = 0.95). This reflects ET_B effect was overall uniform across all heart structures, albeit minor regional-dependent impact might be present in RV