Li Ning, Csepe Thomas A, Hansen Brian J, Dobrzynski Halina, Higgins Robert S D, Kilic Ahmet, Mohler Peter J, Janssen Paul M L, Rosen Michael R, Biesiadecki Brandon J, Fedorov Vadim V
From 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.).
Circ Arrhythm Electrophysiol. 2015 Oct;8(5):1219-27. doi: 10.1161/CIRCEP.115.003070. Epub 2015 Aug 24.
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.
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.
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.
超极化激活电流（If）在窦房结（SAN）起搏中起重要作用。令人惊讶的是，人类窦房结中超极化激活环核苷酸门控（HCN）通道的分布仅在mRNA水平进行过研究。我们的目的是确定HCN蛋白在人类窦房结和不同心房区域的表达模式。
从手术室中停搏的衰竭（n = 5）和非衰竭（n = 9）人类心脏中分离出完整的窦房结复合体。三维壁内窦房结结构被确定为围绕窦房结动脉的纤维化致密区域，在Masson三色染色和免疫染色的冰冻切片中可见连接蛋白43阴性的起搏心肌细胞。使用16G活检针从相邻的冷冻窦房结组织块中精确分离出窦房结蛋白。通过连接蛋白43免疫印迹确认窦房结蛋白的纯度。在窦房结中检测到所有3种HCN亚型蛋白。HCN1主要分布在人类窦房结中，窦房结与右心房的表达比值为125.1±40.2（n = 12）。HCN2和HCN4在窦房结中的表达水平高于心房，窦房结与右心房的比值分别为6.1±0.9和4.6±0.6（n = 12）。
这是第一项通过分离纯起搏窦房结组织对人类窦房结进行精确三维分子定位的研究。所有3种心脏HCN亚型在窦房结中的表达均高于心房。HCN1几乎仅在窦房结中表达，强调了其作为人类窦房结新的特异性分子标志物以及作为旨在改变窦性心律的特定治疗潜在靶点的效用。
