Location of the RN, CN and inferior extension
Serial sections (a total of 80 groups) were chosen from an adult rat and revealed the most integrated conduction system. The sequence of sections began from the anterior septum to the posterior septum. The structure of the RN roughly presented from the 1st section to the 37th group of sections neighboring the aorta above the His bundle. Further analysis was performed on the 13thgroup of sections, shown in Figure 2A-D, which presented the main body of the RN and showed its positional relationship with the His bundle. Masson’s trichrome staining showed that the PB tissue (light purple) was surrounded by fibrous tissue (blue) on the ventricular side (Figure 2A). AChE expression was positive on the atrial side over the tricuspid ring, suggesting the distribution of RN (Figure 2B, arrow). Figure 2C highlights the absence of Cx43 expression (green) in both the atrial and ventricular regions, while the expression of AChE and HCN4 was observed in Cx43-negative regions. Figure 2D shows that Cx45 expression was abundant in the PB and the areas where Cx43 expression was absent on the atrial side. Cx40 was expressed only in PB tissues.
The structure of the CN roughly presented from the 38th section to 53rd group of sections. Figure 2E-H represents the 39th group of serial sections at the level coincident with the CN. The CN is considered to be the start of the PB.10 Masson’s trichrome staining showed the transit of all AVN tissues nearly to the atrial side (Figure 2E). AChE-positive staining was present mostly in the area of the CN, and little could be seen above the CN, which suggests the end of the RN (Figure 2F, arrow). At this level, Cx43 and HCN4 double-labeling showed the greatest absence of Cx43 expression limited to the AVN area where HCN4 expression was positive (Figure 2G). Little of the HCN4-positive area on the atrial side coincided with the AChE-positive site. As shown in Figure 2H, Cx45 was found to be abundant in the AVN, although there was slight cross-reactivity with the connective tissue on the ventricular side. In addition, a punctate distribution pattern of Cx40 expression showed a disappearing trend (elliptical area).
The structure of the inferior extension of the AVN roughly presented from 54th group of sections to the 80th group of sections. The 79th group of sections was near the coronary sinus level. Masson’s trichrome staining showed the inferior extension of the AVN located on the atrial side above the tricuspid annules (Figure 2I). AChE staining was limited to the INE area (Figure 2J). The volume of the inferior extension region also decreased moving away from the CN. Cx43 expression was absent in the inferior extension where HCN4 expression was positive (Figure 3K). Cx45 expression was relatively more abundant in the INE than in other atrial areas (Figure 3L, a slight fold in the section). No Cx40 expression was observed in the inferior extension. The inferior extension divided into right and left extensions at this level (Figure 3K).