Connexin 43 expression delineates two discrete pathways in the human atrioventricular junction

Abstract

Gap junction expression has been studied in the atrioventricular junction (AVJ) of many species, however, their distribution in the human AVJ is unknown. The AVJ expression of the gap junction protein connexin 43 (Cx43) is species dependent; therefore we investigated its distribution in the human AVJ. Using Masson trichrome histology, we reconstructed the AVJ of three normal human hearts and one with dilated cardiomyopathy in three dimensions. Cx43 was immunolabeled with vimentin and alpha-actinin to determine the cellular origin of Cx43 and was quantified in the following structures: interatrial septum (IAS), His bundle, compact node (CN), lower nodal bundle (LNB), leftward and rightward nodal extensions (LE and RE), and inferior, endocardial, and left-sided transitional cells. Histology revealed two nodal extensions in three of four hearts. Cx43 was found in the myocytes, but not fibroblasts, of the AVJ. LE and CN Cx43 was lower than the IAS (P < 0.05) and the RE, LNB, and His all expressed Cx43 similarly, with approximately half of IAS expression (RE: 44 +/- 36%; LNB: 50 +/- 26%; His: 48 +/- 12%, P = NS compared with IAS). Cx43 levels in transitional cells were similar to the IAS (P = not significant). Cx43 was found in myocytes of the human AVJ, and its expression pattern delineates two separate continuous structures: one consists of the LE and CN with little Cx43, and the other consists of the His, LNB, and RE expressing approximately half the Cx43 of the IAS. The differential Cx43 expression may provide each structure with unique conduction properties, contributing to arrhythmias arising from the AVJ.

Fig. 1

Dissection of human atrioventricular junction (AVJ) and creation of a three-dimensional (3D) reconstruction. A: Dissected AV junctional preparation with anatomical landmarks labeled. B: High-resolution image of the boxed area in A, with locations of sections stained with Masson trichrome marked. Also the triangle of Koch is outlined, which is bounded by the tendon of Todaro (TT), the coronary sinus (CS), and the septal leaflet of the tricuspid valve (TV). C: Masson trichrome staining of section marked by a red line in B, with different tissue areas outlined for 3D reconstruction. D: Outlines of the conduction system of all sections aligned in three dimensions. Red outline corresponds to the AV node outlined in C. E: Outlines of the conduction system lofted to create a 3D mesh. F: The 3D mesh in E rendered to approximate the 3D volume. AVN, AV node; CFB, central fibrous body; FO, fossa ovalis; IAS, intraatrial septum; LE: leftward extension; RE, rightward extension; VS, ventricular septum; S,I,P,A, superior, inferior, posterior, anterior orientation.

Fig. 2

Cx43 density in the nodal extensions. A: Masson trichrome stain of the nodal extensions. Outlined area surrounding the nodal extensions corresponds to immunohistochemistry shown in panels B and C. B: Immunohistochemistry of the nodal extensions showing α-actinin in red, vimentin in blue, and Cx43 in green. C: Cx43 expression in the nodal extensions. D—F: Higher magnification of Cx43, vimentin, and α-actinin expression in leftward extension (D), the rightward extension, and the inferior transitional cells (E,F). G: Three-dimensional reconstruction of the atrioventricular junction (AVJ) split open at the plane of section shown in panels A—C. CFB, central fibrous body; IAS, interatrial septum; LE, leftward extension; RE, rightward extension; VS, ventricular septum; P,A, posterior—anterior orientation.

Fig. 3

Cx43 density in the atrioventricular node (AVN). A: Masson trichrome stain of the AVN. Outlined area surrounding the AVN corresponds to immunohistochemistry shown in panels B and C. B: Immunohistochemistry of the AVN showing α-actinin in red, vimentin in blue, and Cx43 in green. C: Cx43 expression in the AVN. D—G: Higher magnification of Cx43, vimentin, and α-actinin expression in various areas of the AVN region. H: Three-dimensional reconstruction of the AVJ split open at the plane of section shown in A—C. CFB, central fibrous body; IAS, interatrial septum; LNB, lower nodal bundle; VS, ventricular septum; P,A, posterior—anterior orientation.

Fig. 4

Cx43 density in the His bundle. A: Masson trichrome stain of the His bundle. Outlined area surrounding the His bundle corresponds to immunohistochemistry shown in B and C. B: Immunohistochemistry of the His bundle showing α-actinin in red, vimentin in blue, and Cx43 in green. C: Cx43 expression in the His bundle. D: Higher magnification of Cx43, vimentin, and α-actinin expression in the His bundle. E: Three-dimensional reconstruction of the atrioventricular junction (AVJ), which was split open at the plane of section shown in panels A—C. CFB, central fibrous body; IAS, interatrial septum; VS, ventricular septum; P,A, posterior—anterior orientation.

Fig. 5

Cellular Expression of Cx43. A: Maximum projection image of Cx43 (green) α-actinin (red) and vimentin (blue) staining in the lower nodal bundle (LNB). B: Colocalization of Cx43 and α-actinin, showing that voxels of high Cx43 intensity also have a high α-actinin signal. C: Colocalization of Cx43 and vimentin, showing that voxels of high Cx43 intensity have no significant vimentin signal. D,E: Data similar to A—C for the compact node (CN). See text for details.

Fig. 6

Cx43 density in transitional cells in the atrioventricular junction (AVJ). Cx43 density in the endocardial (endo), left-sided, and inferior transitional cells. All densities are normalized to the Cx43 density of the interatrial septum (IAS). DCM, dilated cardiomyopathy.

Fig. 7

Cx43 density in the conduction system of the atrioventricular junction (AVJ). All densities are normalized to the Cx43 density of the interatrial septum (IAS). CN, compact atrioventricular node; DCM, dilated cardiomyopathy; LNB, lower nodal bundle; LE, leftward extension; RE, rightward extension.

Fig. 8

Three-dimensional reconstruction of the atrioventricular junction (AVJ) conduction system. A—C: Endocardial view of the conduction system of each normal heart. Left side of each panel displays the connective tissue and blood vessels surrounding the conduction system, as well as the location of the conduction system within the triangle of Koch of each preparation. Right side of each panel shows the conduction system and the three planes where Cx43 was quantified. D: Conduction system reconstruction of the heart with dilated cardiomyopathy. Left side of panel displays the same endocardial view as shown in A—C. Middle of panel shows the conduction system rotated 90 degrees to more clearly show leftward extension. CFB, central fibrous body; IAS, interatrial septum; TT, tendon of Todaro; VS, ventricular septum; A—P, S—I, D—V, anterior—posterior, superior—inferior, and dorsal—ventral orientations.