University Hospital Munich, Department of Medicine I, Ludwig Maximilian University Munich; Walter Brendel Center of Experimental Medicine, Ludwig Maximilian University Munich; German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance;
University Hospital Munich, Department of Medicine I, Ludwig Maximilian University Munich; Walter Brendel Center of Experimental Medicine, Ludwig Maximilian University Munich.
J Vis Exp. 2020 Dec 22(166). doi: 10.3791/62058.
The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging. The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment. Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers. Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.
窦房结（SAN）起搏细胞产生的生理电信号通过传导系统传导，该系统包括房室结（AVN），从而使整个心脏兴奋和收缩。SAN 或 AVN 的任何功能障碍都会导致心律失常，这表明它们在电生理学和心律失常发生中起着基本作用。小鼠模型广泛用于心律失常研究，但对 SAN 和 AVN 的具体研究仍然具有挑战性。SAN 位于心耳末端嵴与上腔静脉的交界处，AVN 位于 Koch 三角的顶点，由冠状窦口、三尖瓣环和 Todaro 肌腱形成。然而，由于 SAN 和 AVN 体积小，常规组织学观察仍然具有挑战性，并且不允许在其 3D 环境中研究 SAN 和 AVN。在这里，我们描述了一种全组织免疫荧光方法，该方法可局部观察标记的小鼠 SAN 和 AVN。全组织免疫荧光染色适用于较小的组织切片，无需手动切片。为此，需要解剖小鼠心脏，去除不需要的组织，然后进行固定、通透和封闭。SAN 和 AVN 中的传导系统细胞用抗 HCN4 抗体染色。共聚焦激光扫描显微镜和图像处理允许区分节细胞和工作心肌细胞，并明确定位 SAN 和 AVN。此外，还可以结合其他抗体来标记其他细胞类型，如神经纤维。与传统免疫组织化学相比，全组织免疫荧光染色保留了心脏传导系统的解剖完整性，从而允许对 AVN 进行研究；特别是对其解剖结构及其与周围工作心肌和非心肌细胞的相互作用进行研究。
