title: Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node.
abstract: Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I(f) current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.
authors.affiliation: University of Milano, Department of Biomolecular Sciences and Biotechnology, Laboratory of Molecular Physiology and Neurobiology, via Celoria 26, 20133 Milano, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Brioschi
authors.forename: Stefano
authors.initials: S
authors.lastname: Micheloni
authors.forename: James O
authors.initials: JO
authors.lastname: Tellez
authors.forename: Giuliano
authors.initials: G
authors.lastname: Pisoni
authors.forename: Renato
authors.initials: R
authors.lastname: Longhi
authors.forename: Paolo
authors.initials: P
authors.lastname: Moroni
authors.forename: Rudi
authors.initials: R
authors.lastname: Billeter
authors.forename: Andrea
authors.initials: A
authors.lastname: Barbuti
authors.forename: Halina
authors.initials: H
authors.lastname: Dobrzynski
authors.forename: Mark R
authors.initials: MR
authors.lastname: Boyett
authors.forename: Dario
authors.initials: D
authors.lastname: DiFrancesco
authors.forename: Mirko
authors.initials: M
authors.lastname: Baruscotti
mesh_terms: D000818:Animals; D001683:Biological Clocks; D015153:Blotting, Western; D002469:Cell Separation; D018031:Connexin 43 / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D012849:Sinoatrial Node / Q000166:cytology / Q000378:metabolism*
chemical_list: D018031:Connexin 43; D054815:Cyclic Nucleotide-Gated Cation Channels; D012333:RNA, Messenger