Files in this Data Supplement:

• Supplemental Methods - (Word) (80.5 KB)
• Figure I - (TIFF) (17.5 MB) Colocalization of Na_v1.3 and neurofilament 160 in the penetrating AV bundle. High-magnification images of double labelling of Na_v1.3 and neurofilament 160 in the penetrating AV bundle from three preparations. Left panels, Na_v1.3 labelling (green). Middle panels, neurofilament 160 labelling (red). Right panels, superimposed images of Na_v1.3 and neurofilament 160 labelling; where the Na_v1.3 and neurofilament 160 labelling is colocalized, there is a yellow color. Scale bars, 50 μm (top) and 100 μm (middle and bottom).
• Figure II - (TIFF) (7.62 MB) Semi-quantitative assessment of Na_v1.5 expression in the different regions of the AV node. The relative level of Na_v1.5 expression was determined in a semi-quantitative manner with Scion image (release: Alpha 4.0.3.2). In sections through the AVN the average pixel intensity of different regions was measured. In order to compare intensities of different regions, all measured intensities in one section were expressed as a percentage of the highest intensity value (from the ventricular myocardium). Means±SEM shown (number of hearts, 4; number of sections, 20). Significant differences between regions are shown as numbers above the bars (P<0.05, one-way ANOVA).
• Figure III - (TIFF) (13.7 MB) Specificity of Na_v1.5 antibody. A, Western blot of Na_v1.5 in rat ventricular tissue. A band at the correct molecular weight was identified by the antibody as shown in the left hand lane. The band was absent in the right hand lane when the Na_v1.5 antibody was preincubated with the antigenic peptide (thus confirming the specificity of the antibody). B, immunolabelling of HEK-293 cells transiently expressed with Na_v1.5. Left, GFP fluorescence (green); middle, Na_v1.5 labelling (red); right, superimposition of GFP and Na_v1.5 signals (nuclei stained with DAPI - blue). DAPI staining shows that there were many cells in the field of view, but only the transfected cell (expressing GFP and, therefore, Na_v1.5) showed Na_v1.5 labelling.
• Figure IV - (TIFF) (8.61 MB) Labelling of Cx40 (green) in the penetrating bundle (outlined by yellow dashed line). Two groups of nodal myocytes can be identified: Cx40-negative myocytes (upper part) and Cx40-positive myocytes (lower part).
• Figure V - (TIFF) (24.7 MB) Expression of Cx40, Cx43, HCN4 and Na_v1.5 in the left bundle branch. A, section labelled for Cx40 (green). B, adjacent section labelled for HCN4 (green) and Cx43 (red). C, adjacent section labelled for Na_v1.5 (green) and Cx43 (red). Yellow ringed areas highlight the left bundle branch. VM, ventricular myocardium. Scale bars, 200 μm.
• Figure VI - (TIFF) (18.7 MB) Expression of Cx43 and HCN4 in the AV ring bundle. A, Masson's trichrome stained section (cut along the sagittal plane; the position of the cutting plane is shown by the dashed line in Figure 1B). B, high magnification image of the framed area in A. C, double labelling of Cx43 (red) and HCN4 (green) in the AV ring bundle on the opposite side of the mitral valve to the AVN. D, double labelling of Cx43 (red) and HCN4 (green) in the common bundle (and transitional zone). C and D correspond to the framed areas in B. AS indicates atrial septum; CT, crista terminalis; LA, left atrium; LV, left ventricle; MV, mitral valve; RA, right atrium; RV, right ventricle; SVC, superior vena cava; TV, tricuspid valve; and VS, ventricular septum.
• Figure VII - (TIFF) (16.3 MB) Expression of Na_v1.1 and Na_v1.5 in the AV ring bundle. A, B, double labelling of Cx43 (red) and Na_v1.1 (green). C, D, double labelling of Cx43 (red) and Na_v1.5 (green). A, C, AV ring bundle on the opposite side of the mitral valve to the AVN. B, D, common bundle (and the transitional zone). A and C correspond to framed area C in Figure VI and B and D correspond to framed area D in Figure VI. Scale bars, 200 μm.
• Figure VIII - (TIFF) (23.5 MB) Masson's trichrome labelled sections at the 14 levels shown in Figure 1A. AM indicates atrial myocardium; Ao, aorta; AoV, aortic valve; TV, tricuspid valve; and VM, ventricular myocardium. Scale bars, 200 μm.