Fig 2: Intracellular Microelectrode Recording From a Representative Right Atrial Cell(A) Excerpt of the intracellular recording in control conditions (black) and in the presence of 200 nM S100 calcium-binding protein B–positive (S100B) (blue). (B) Variability in inter-action potential (AP) cycle lengths and the mean inter-AP cycle length for all APs occurring in the time series in control and in the presence of S100B. In both, the dotted line indicates the mean interval, and a running average for the time series is shown. (C) A histogram of the distribution of AP firing interval under control conditions, in the presence of 200 nM S100B, and after 40 minutes of washout. (D) Poincaré analysis of the time series, with the insets displaying a magnified version of the Poincaré plots for control and washout conditions. The best fit line of all the points is given by the equation y = 1.0x + 1.63, R2 = 1.0, indicating the self-similarity among the points in the time series, although their absolute magnitudes differ.

Fig 3: 2-Dimensional Images of a Whole-Mount Preparation of SAN Tissue With Triple Immunolabeling for S100B, HCN4, and TH or VAChT(A to C) Two-dimensional images of SAN tissue with triple immunolabeling for S100B+ cells (cyan), HCN4-immunoreactive pacemaker cells (red), and TH+ adrenergic fibers (green) illustrate anatomical interactions between pacemaker cells, adrenergic nerves, and interstitial cells. (D to F) Two-dimensional images of SAN tissue with triple immunolabeling for S100B+ cells (cyan), HCN4 immunoreactive pacemaker cells (red), and VAChT+ cholinergic fibers (green) illustrate anatomical relations between pacemaker cells, adrenergic nerves, and interstitial cells. Pink stars in any panel indicate the nuclei of peripheral glial cells. S100B+ spicula extended from “octopus”-like cells in A, C, and E ended on TH+ varicosities (A and C) or on VAChT+ varicosities (E). Yellow arrows indicate the S100B+ “endfeet.” An adrenergic neurite on A, indicated by a white star, overlaps with the spiculum of an “octopus”-like S100Bþ cell. An amoeboid-like S100B+ cell in the upper right corner of C receives adrenergic innervation. Images on C (adrenergic nerves) and D (cholinergic nerves) illustrate uneven innervation of S100B+ cells. (F) Composite point of contact between 3 cells: one S100B-immunoreactive cell, an HCN4-immunoreactive cell, and fibers from the neuronal plexus. A white arrow indicates the region where cellular extensions from an intertwined couple of S100B+ cells, an HCN4+ pacemaker cell, and a cholinergic neurite co-localize within 1 µm of each other. A white star indicates the point of contact between an S100B+ cell and a cholinergic nerve, whereas a white triangle marks the point of contact between a cholinergic nerve and an HCN4+ pacemaker cell. Abbreviations as in Figures 1, 3, and 4.

Fig 4: 2-Dimensional Images of S100B+/GFAP- Interstitial Cells and PGCs Embedded Within the HCN4+ MeshworkPink arrows indicate S100B+/GFAP- interstitial cells, and yellow arrows indicate S100B+/GFAP+ PGCs. (A to D) S100B+/GFAP- interstitial cells (cyan) between, and in close proximity to, HCN4+ pacemaker cells (red). Higher levels of GFAP (green) than S100B were detected in the extensions of PGCs. (E) A 2-dimensional image of the radiating branches of adrenergic TH+ (green) fibers as well as S100B+ interstitial cells (cyan), among HCN4-immunoreactive pacemaker cells (red). (F) A 2-dimensional image of the radiating branches of cholinergic VAChT+ (green) fibers and S100B+ interstitial cells (cyan), among HCN4-immunoreactive pacemaker cells (red). Abbreviations as in Figures 2 and 4.

Fig 5: Variability in the Number of Detected S100B+ Interstitial Cells in the Head, Body, and Tail of the SAN(A) Area near the root of the SVC, known as a “head,” of the HCN4+ meshwork of pacemaker cells (red), from 3 different SAN preparations. (B) S100B cell populations in the “body” of the SAN between the SVC and the IVC. (C) Area close to the IVC known as the “tail” of the SAN. In all 3 panels, the upper images illustrate examples of SANs in which >110 S100B+ cells were identified, the middle images illustrate meshworks exhibiting ~60 S100B+ cells, and the lower images show SANs with <25 S100B+ cells. Broken yellow lines indicate the relative position of the CT in each frame. Abbreviations as in Figures 1 and 4.