Septation of the Intrapericardial Arterial Trunks in the Early Human Embryonic Heart

Abstract

Background: Outflow tract (OFT) septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart.

Methods: Serial sections of 27 human embryonic hearts from Carnegie stage (CS) 10 to CS19 were immunohistochemically stained with antibodies against α-smooth muscle actin (α-SMA) and myosin heavy chain.

Results: At CS10-CS11, the OFT wall was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16, the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19, the bilateral cushions were fused to septate the myocardial portion of the OFT.

Conclusions: Data suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopulmonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.

Keywords: Aortopulmonary Septum; Human Embryonic Heart; Immunohistochemistry; Outflow Tract; Outflow Tract Cushion.

Figure 1

The aortic sac and OFT of the human embryonic heart at CS11. The aortic sac was located at the ventral pharyngeal mesenchyme, the α-SMA-positive (a) and MHC-positive (b) OFT myocardium extended to the reflection of the OFT and the pericardial dorsal wall (arrows). No mesenchymal cells were observed in the cardiac jelly of the OFT. Immunohistochemical staining. Scale bar: 50 μm. e: Endothelium; m: Myocardium; SMA: Smooth muscle actin; MHC: Myosin heavy chain; AS: Aortic sac; OFT: Outflow tract; PC: Pericardial cavity; CS: Carnegie stage.

Figure 2

The aortic sac and OFT of the human embryonic heart at CS12 (a) and CS13 (b–d). The aortic sac was situated in the pharyngeal mesenchyme (a). An α-SMA-negative portion was observed at the distal pole of the OFT wall (b, arrowheads). The cardiac jelly of the OFT was filled with mesenchymal cells (c). In the distal portion of the OFT, some of the mesenchymal cells were α-SMA positive (a and b, arrows). In the proximal portion of the OFT, the mesenchymal cells were α-SMA negative (d). (a, b and d) immunohistochemical staining with α-SMA; and (c) H and E staining. (a) Scale bar: 100 μm; and (b–d) scale bar: 200 μm. SMA: Smooth muscle actin; PC: Pericardial cavity; AS: Aortic sac; OFT: Outflow tract; P-OFT: The proximal part of the outflow tract; CS: Carnegie stage.

Figure 3

The aortic sac and OFT of the human embryonic heart at CS14. (a–e) The OFT contained α-SMA- and MHC-negative portion and α-SMA- and MHC-positive portion. (f–h) The number of mesenchymal cells increased in the OFT cushion and α-SMA-positive cells were observed throughout the cushion. (h) Two α-SMA-negative cell masses were observed between the OFT cushions (asterisks). In left view of the heart (e), the myocardium is labeled in gray. The nonmyocardial OFT is labeled in pink. The fourth aortic arch arteries and the aortic sac are noted in red. The sixth aortic arch arteries are noted in brown. (a, c, g, and h) IHC staining with α-SMA; (b and d) IHC staining with MHC; and (f) H and E staining. (a–d) Scale bar: 200 μm; and (f–h) scale bar: 50 μm. The number 4 and 6 indicate the fourth and sixth aortic arch arteries, respectively. sep: Septal cushion; par: Parietal cushion; SMA: Smooth muscle actin; MHC: Myosin heavy chain; IHC: Immunohistochemical; H and E: Hematoxylin and eosin; PC: Pericardial cavity; AS: Aortic sac; A: Atrium; OFT: Outflow tract; LA: Left atrium; LV: Left ventricle; D-OFT: The distal part of the outflow tract; P-OFT: The proximal part of the outflow tract; CS: Carnegie stage.

Figure 4

The aortic sac and OFT of the human embryonic heart at CS15. The aortic sac between the fourth and sixth aortic arch arteries degenerated (a). The MHC-negative area was located in the ventral and dorsal walls of the distal portion of the OFT (a, b, g and h). In the ventral wall, MHC expression was negative in the right wall (b, arrow). When the OFT extended dorsally and caudally, the right wall became positive for MHC (d, arrow). α-SMA-positive cells increased in the cardiac jelly of the right wall (c and e, asterisks). The cushions contained numerous α-SMA-positive cells (f). In a and h, the myocardium is labeled in gray; the nonmyocardial OFT is in pink; the fourth aortic arch arteries and the aortic sac are in red; and the sixth aortic arch arteries are in brown. A shows the left view of the heart. H shows the ventral view. (b, d, and g) IHC staining with MHC; (c, e, and f) IHC staining with α-SMA. (b–e) Scale bar: 50 μm; and (f and g) scale bar: 100 μm. The number 4 and 6 indicate the fourth and sixth aortic arch arteries, respectively. sep: Septal cushion; par: Parietal cushion; SMA: Smooth muscle actin; MHC: Myosin heavy chain; IHC: Immunohistochemical; AS: Aortic sac; OFT: Outflow tract; RV: Right ventricle; CS: Carnegie stage.

Figure 5

The OFT of the human embryonic heart at CS16 (a–e) and CS17 (f and g). The α-SMA-positive aortopulmonary septum formed and fused incompletely with the two cushions, separately (a–e). As a result, the nonmyocardial portion of the OFT was divided into the aorta and pulmonary trunk (a-e). The aortopulmonary foramen was observed at CS16 (c, arrow). At the root of the artery, the valve anlage began to emerge and was contiguous with the OFT cushion (c and e). The myocardial OFT and its cushion became shortened (b–e). The two cushions were not fused (c–e). At CS17, the aortopulmonary foramen was not observed (f and g, asterisks). In ventral view of the heart (e), the myocardium is labeled in gray; the nonmyocardial OFT is in pink; the aorta is in red; the sixth aortic arch arteries are in brown; the aortopulmonary septum is in yellow; the valve anlage is in blue; and the cushion is in green. (a, c, and d) IHC staining with α-SMA; (b) IHC staining with MHC; (f and g) H and E staining. (a–c and g) Scale bar: 100 μm; and (d and f) Scale bar: 200 μm. Ao: Ascending aorta; sep: Septal cushion; par: Parietal cushion; SMA: Smooth muscle actin; MHC: Myosin heavy chain; IHC: Immunohistochemical; H and E: Hematoxylin and eosin; PT: Pulmonary trunk; APS: Aortopulmonary septum; V: Valvar primordium; I: Intercalated cushion; RV: Right ventricle; LV: Left ventricle; RA: Right atrium; LA: Left atrium; OFT: Outflow tract; CS: Carnegie stage.

Figure 6

The OFT of the human embryonic heart at CS19. In the aorta and the pulmonary trunk wall, α-SMA-positive cells surrounded the endothelium (a–c). MHC expression was positive in the OFT wall (d–f). The arterial valves were clearly observed at the roots of the aorta and pulmonary trunk (a-e). Below the valve level, the two cushions began to undergo fusion (b, arrow). Even after the OFT extended to the right ventricle, the cushions were not fused yet (c and f, arrows). (a–c) IHC staining with α-SMA; (d–f) IHC staining with MHC. (a–f) Scale bar: 200 μm. Ao: Aorta; SMA: Smooth muscle actin; MHC: Myosin heavy chain; IHC: Immunohistochemical; H and E: Hematoxylin and eosin; PC: Pericardial cavity; PT: Pulmonary trunk; SV: Semilunar valve; OFT: Outflow tract; CS: Carnegie stage.