Molecular Mapping of Sinoatrial Node HCN Channel Expression in the Human Heart

Abstract

Background The hyperpolarization-activated current, If, plays an important role in sinoatrial node (SAN) pacemaking. Surprisingly, the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels in human SAN has only been investigated at the mRNA level. Our aim was to define the expression pattern of HCN proteins in human SAN and different atrial regions. Methods and Results Entire SAN complexes were isolated from failing (n=5) and non-failing (n=9) human hearts cardioplegically-arrested in the operating room. Three dimensional intramural SAN structure was identified as the fibrotic compact region around the SAN artery with Connexin43-negative pacemaker cardiomyocytes visualized in Masson’s trichrome and immunostained cryosections. SAN protein was precisely isolated from the adjacent frozen SAN tissue blocks using a 16G biopsy needle. The purity of the SAN protein was confirmed by Connexin43 immunoblot. All three HCN isoform proteins were detected in SAN. HCN1 was predominantly distributed in the human SAN with a 125.1±40.2 (n=12) expression ratio of SAN to right atrium (RA). HCN2 and HCN4 expression levels were higher in SAN than atria with SAN to RA ratios of 6.1±0.9 and 4.6±0.6 (n=12), respectively. Conclusions This is the first study to conduct precise 3D molecular mapping of the human SAN by isolating pure pacemaker SAN tissue. All three cardiac HCN isoforms had higher expression in the SAN than the atria. HCN1 was almost exclusively expressed in SAN, emphasizing its utility as a new specific molecular marker of the human SAN and as a potential target of specific treatments intended to modify sinus rhythm.

Redundant and diverse intranodal pacemakers and conduction pathways protect the human sinoatrial node from failure

“…Masson’s trichrome staining distinguished the SAN from neighboring atria as a dense fibrotic (45.8 ± 4.4% fibrosis) and cardiomyocyte compact region around the SAN artery, which was validated by negative immunostaining for the gap junction protein connexin 43 (Cx43) (Fig. 1B) (2, 19). The SAN was almost entirely structurally insulated from the surrounding atria by a border composed of fibrosis, fat, and discontinuous myofibers except in functionally defined SACP regions, where several intramural continuous branching myofiber tracts (100 to 400 μm thick and 2 to 3 mm long) of transitional cells were defined (Fig.…”

“…As our optical mapping consistently demonstrated a high sensitivity of the central SAN pacemaker to adenosine’s bradycardic effect, we tested the hypothesis that expression of A1R and GIRK1/4, the main cardiac proteins responsible for adenosine’s negative chronotropic effects, was higher in the human SAN center than the adjacent atria (19, 29). Our methods to extract pure SAN protein made it possible to compare the protein profile among the three (head, center, and tail) intranodal pacemaker regions (Fig.…”

Human sinoatrial node structure: 3D microanatomy of sinoatrial conduction pathways

“…In each 2D histology section, myocardial tissue was delineated from fat, blood vessels and fibrosis for subsequent 3D analysis ( Figure 5 ). Furthermore, histology and immunostaining images were used to identify and delineate the SAN pacemaker tissue from the surrounding right atria based on positive (atrial myocardium) and negative (SAN) Cx43 expression, distinct cell morphology, cell diameter and percent tissue fibrosis as previously described by our group and other human SAN studies ( Figures 7-9 ) 3, 7, 9, 28, 31, 40, 41 . SACP structure was identified as tracts of myofibers with transitional cells in the SAN border that merge with atrial myobundles and had distinctly different fiber orientations compared to the main SAN and atrial fiber orientations.…”

Two‐Pore K + Channel TREK‐1 Regulates Sinoatrial Node Membrane Excitability

“…Immunostaining of tissue sections was performed on 1 nondiseased and 1 diseased heart, as described 19. The study was approved by the Ohio State University institutional review board, and participants gave informed consent.…”