Synchronized Cardiac Impulses Emerge from Heterogeneous Local Calcium Signals Within and Among Cells of Pacemaker Tissue

National Institute On Aging

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Abstract

1) Our results add additional insight into the structural and functional complexity of SAN tissue. 1A) Our data show previously unreported topography of the neuronal and glial plexuses within SAN tissue and illustrate their interactions with the pacemaker cell meshwork. Triple immunolabelling of whole mount SAN tissue with antibodies against choline acetyltransferase (ChAT) or vesicular acetylcholine transporter (VAChT) (cholinergic neural fibers), tyrosine hydroxylase (TH) (adrenergic neural fibers) and HCN4 (pacemaker cells) revealed the neuronal plexus rich in both cholinergic and adrenergic nerve fibers with a high number of varicosities. TH and VAChT immunoreactive axons diverged to smaller branches to innervate individual cells or cellular units within the HCN4-positive meshwork. 3D-images of the TH and VAChT immunoreactive neuronal plexus displayed an area within the HCN4+-immunoreactive cellular meshwork with the predominant cholinergic innervation. 1B) Glial network was double immunolabeled for glial markers GFAP, intermediate filament protein and S100B, a Ca2+ binding protein containing 2 EF-hand Ca2+-binding motifs, characterized by diverse functions. We showed that the S100B+/GFAP+ glial cells in the SAN are interconnected with GFAP-immunoreactive branches, creating a web-like regular network within HCN4-immunoreactive meshwork located between IVC and SVC, which extended beyond the SAN area into the crista terminalis on one side and towards the septum on the other side. Glial cells are characterized by a triangular soma shape and are positioned along the neuronal fibers. 1C) Interestingly, this whole-mount tissue immunolabeling and 3-D tiling imaging approach aided us in uncovering of a new previously undescribed network of new type of cells within the sinus node, these cells are S100B+/GFAP- and display phenotype distinct from neurons and glial cells but similar to interstitial cells described in other tissues. We observed complex and frequent anatomical interaction between the S100b+- cells and pacemaker cells. They often display long branching dendritic spine-like projections with multiple ultrathin processes enwrapping pacemaker cells, and often have large end foot-like structures adhering to pacemaker cells suggesting the presence of a dynamic functional interaction between these cell types. 2) Heterogeneity of local Ca2+ signaling characteristics among cells translates into several new patterns of intercellular communication within the HCN4 pacemaker meshwork. Oscillatory Ca2+ signals, including both those occurring locally, i.e., spontaneous LCRs, and those induced by APs i.e., APCTs that occur within clusters of cells comprising the HCN4 meshwork are markedly heterogeneous. Communication between pacemaker cells rests on their capacity to change their rhythmic activity to interpret different messages. We showed that in the SAN this ability also depends on regulation of the extracellular Ca2+ concentration by S100B released from S100B+ cells. We employed nanomolar concentration of extracellular S100B, a Ca2+-binding protein, to disrupt Ca2+ homeostasis in the isolated SAN tissue. Adding S100b, to the extracellular space, markedly slows down the rhythmic activity. These results indicate that S100B+ cells play a role in modulation of the pacemaker firing pattern.