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Published on: December 2, 2016
Molecular Mapping of Sinoatrial Node HCN Channel Expression in the Human Heart
Ning Li1, Thomas A Csepe1, Brian J Hansen1
1From the Department of Physiology & Cell Biology and Dorothy M. Davis Heart & Lung Research Institute (N.L., T.A.C., B.J.H., P.J.M., P.M.L.J., B.J.B., V.V.F.), Department of Surgery and Dorothy M. Davis Heart & Lung Research Institute (R.S.D.H., A.K.), The Ohio State University Wexner Medical Center, Columbus; Institute of Cardiovascular Sciences, University of Manchester, Manchester, United Kingdom (H.D.); and Departments of Pharmacology and Pediatrics, Columbia University, New York, NY (M.R.R.).
Insights
This study precisely mapped hyperpolarization-activated cyclic nucleotide-gated (HCN) protein expression in the human sinoatrial node (SAN). All three HCN isoforms were found at higher levels in the SAN, with HCN1 being a specific marker for this crucial heart tissue.
Area of Science:
- Cardiology
- Molecular Biology
- Electrophysiology
Background:
- The hyperpolarization-activated current (If) is vital for sinoatrial node (SAN) pacemaking.
- Previous studies only examined hyperpolarization-activated cyclic nucleotide-gated (HCN) channel distribution at the mRNA level in the human SAN.
- The protein expression pattern of HCN channels in the human SAN and atria remained undefined.
Purpose of the Study:
- To precisely define the expression pattern of HCN proteins within the human SAN.
- To compare HCN protein distribution between the human SAN and adjacent atrial regions.
Main Methods:
- Isolation of entire SAN complexes from human hearts (n=14).
- Identification of 3D intramural SAN structure using histological and immunohistochemical techniques.
- Precise isolation of SAN protein using a biopsy needle, confirmed by Connexin 43 immunoblot.
Main Results:
- All three cardiac HCN isoform proteins (HCN1, HCN2, HCN4) were detected in the human SAN.
- HCN1 showed predominant distribution in the SAN (SAN to right atrium ratio: 125.1±40.2).
- HCN2 and HCN4 expression levels were significantly higher in the SAN compared to atria (ratios: 6.1±0.9 and 4.6±0.6, respectively).
Conclusions:
- This study provides the first precise 3D molecular mapping of the human SAN using isolated pacemaker tissue.
- All three cardiac HCN isoforms are upregulated in the human SAN compared to atrial tissue.
- HCN1's exclusive expression in the SAN identifies it as a specific molecular marker and a potential therapeutic target for modulating heart rhythm.
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 nonfailing (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 Connexin 43-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 Connexin 43 immunoblot. All 3 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. HCN2 and HCN4 expression levels were higher in SAN than in atria, with SAN to right atrium 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 3 cardiac HCN isoforms had higher expression in the SAN than in 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.
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