Ultrastructure of primary pacemaking cells in rabbit sino‐atrial node cells indicates limited sarcoplasmic reticulum content

Figures

• 

  Figure 1. The SA Node. Dissection of a SAN from rabbit heart previously fixed by perfusion. The sample is pinned, the ligation at the upper left was used for help in the dissection. The magenta dotted line follows the outline of the SAN upper (top) and lower regions; the cyan dots follow the crista terminalis; the yellow arrows point to two of the almost transparent regions that were embedded and sectioned for EM. Atrial tissue is at the left of the SAN

• 

  Figure 2. Low‐power image of a section across the center of a node area such as indicated by arrows in Figure 1. Dense collagen bands (arrows) separate cell‐rich bands which are also infiltrated by collagen bundles. Connective tissue plus endothelium (right) and mesothelium (left) cover the two surfaces. No obvious ultrastructural differences were observed between cells on the two sides

• 

  Figure 3. A, This image is constructed from a series of low magnification micrographs: occasional discontinuities occur where the matching was not perfect. The outline of an elongated cell is shown in its entirety. Most of the cell surface is isolated from neighboring cell by either a single of a double layer of basal lamina, but close contacts are often present (between green arrows). At the top of the image, the cell ends into a junction connecting the two cells via small actin‐based adhering junctions, such as found at intercalated discs of the working heart (between blue arrows). B, Same cell as in (A). The rough yellow outline enhances the very long and thin shape of the cell, with a branch at the upper end, as shown in the literature for isolated cell. Short green segments indicate the position and approximate length of peripheral couplings

• 

  Figure 4. A, Profile of a typical “dark “cell. The cytoplasm is tightly packed with intermediate filaments running in all directions in addition to few mitochondria (M, small, round objects) and scarce myofibrils. Peripheral couplings are present (between small arrows) but free sarcoplasmic reticulum is rare. On the upper edge, the two adjacent cells are separated by their own independent basal laminae; on the left side, the two cells share a common basal lamina; along the lower edge, the two facing cells are closely apposed, with a separation of ~100 nm. Several sites along the perimeter show densities associated with adhering junctions (AJ). Most of them (at the cell upper edge) allow anchoring to the basal lamina, while occasionally a corresponding structure in an apposed cell established a cell‐to‐cell anchorage (arrow heads). B, In another dark cell, the intermediate filaments are clearly visible because they are mostly parallel to the plane of the section

• 

  Figure 5. A “pale” cell profile shows large apparently empty areas and few intermediate filaments. It has the same content of peripheral couplings (between arrows) and mitochondria (M) as dark cells, very little internal free SR (SR, small arrows) and varied relationship with other cells along its border. In this image, the entire lower region of the cell closely faces a neighboring one. Infrequent views of cells that are included in their entirety within in the section plane (eg, Figure 3) show that the “light” appearance with many apparently empty spaces is maintained over the whole visible region of the cell. We conclude that “light” and “dark” cell profiles do not belong to the same cell

• 

  Figure 6. Both light and dark cells are highly endowed with glycogen. Unlike the rest of the samples, this tissue was treated with potassium ferrocyanide (see methods and 1, 23, 30) that allows visibility of glycogen granules. In A (pale cell), glycogen granules are clumped in “empty spaces,” while in B (dark cells) the glycogen granules are dispersed between neurofilaments

• 

  Figure 7. The jSR forms peripheral couplings at the cell edge (asterisks), connected to the surface membrane by RyRs, visible as small periodic elements in the junctional gap between SR membrane and the plasmalemma. Occasionally, peripheral couplings in two adjacent cells face each other as is the case here. Arrows point to free SR. Cav indicates a group of caveolae

• 

  Figure 8. Images of SAN cells showing free SR in four different cells (A‐D), here highlighted in orange, where the Ca2+ pumping ATPase is located. The free SR elements are located mostly within a peripheral ~0.4 µm‐wide rim of the cell

• 

  Figure 9. SAN cells are not joined by complex intercalated discs, but the extensive areas of close proximity between the cells offer the opportunity for location of communication sites. (A‐D) Arrows indicate sites of small contacts between cells, presumably mini gap junctions. (E) Classical image of an extensive gap junction between two intermediate cells. Such classical gap junctions are extremely rare between primary pacemakers cells, but one is shown in (F)

• 

  Figure 10. Images from two different cells. Caveolae, each appearing as a small‐membrane‐limited balloon are found in extensive clusters, as illustrated here, that are unevenly distributed over some parts of the cell surfaces. We found no clue to the reason for this uneven distribution. Caveolae are not usually associated with active endocytic processes, but a coated endocytic vesicle is rarely associated with a multiple caveolar invagination (arrow in B). Small arrows indicate peripheral couplings