Morphology of mouse sinoatrial node and its expression of NF-160 and HCN4

Abstract

Objective: To explore the morphology and cell component of the sinoatrial node in adult mice and to observe the expressions of neurofilament protein (NF-160) and hyperpolarization-activated cyclic nucleotide regulation of cation channel 4 (HCN4) in sinus node cells. Methods: The right and left atria were collected from 10 adult mice. After paraffin embedding and serial section, these sections underwent Masson trichrome staining and NF-160 immunohistochemical staining, respectively, followed by observation under a light microscope. The right and left atria from another 5 mice were used for immunofluorescence staining using NF-160 and HCN4 antibodies, respectively, followed by observation under a confocal microscope. Results: The mouse sinoatrial node was close to the superior vena cava and below the epicardium. The shape of the mouse sinoatrial node was ovoid or irregular in the horizontal sections. We clearly saw a sinus node artery passing through the sinoatrial node, so the artery can be used as a location marker of the sinoatrial node. Immunofluorescence displayed the expression of NF-160 and HCN4 in both pacemaker cells and transitional cells of the sinoatrial node. Finally, sinoatrial node migrated along with Purkinje fibers of the right atrium. Conclusion: The position of mouse sinoatrial node is more stable. The distal end of sinoatrial node extends to the right subendocardial layer and migrates with Purkinje fibers. In mouse sinoatrial node, both pacemaker cells and transitional cells are regularly distributed, and all have HCN4 and NF-160 expression.

Introduction

The sinoatrial node, a pacing site of sinus rhythm in the cardiac conduction system of mammals, plays an important role in maintaining normal cardiac function. The sinoatrial node contains pacemaker cells, transitional cells and interstitial cells [1,2]. Sinus arrhythmia and sick sinus syndrome are common clinically and their incidences are growing in recent years. These diseases bring great harm to people’s health, and their pathogenesis has not been fully clear yet. With the development of clinical diagnosis and treatment, and due to much attention on the relation between cardiac conduction system and cardiac electrophysiological characteristics, it is believed that sinus arrhythmia is caused by abnormal impulse and/or conduction from the sinoatrial node [3]. It is reported that fatty degeneration, amyloid degeneration, inflammatory infiltration, fibrosis, calcium deposition and other pathological changes were identified in patients’ sinoatrial node [4,5]. This demonstrates that abnormal changes in the tissue and structure of the sinoatrial node may cause the abnormal impulse and/or abnormal conduction. Therefore, the study on the tissue and structure of the sinoatrial node can provide a basis for exploring the pathogenesis of sinus arrhythmia. Since the cardiac structure of mice is similar to that of human, in this study mouse hearts were used as samples. We identified the position, cell component, fine structure and pacing-channel distribution of the sinoatrial node by Masson trichrome staining, neurofilament protein (NF-160) immunohistochemical staining and hyperpolarization-activated cyclic nucleotide regulation of cation channel 4 (HCN4) immunofluorescence staining in this study, providing a basis for studies on cardiac conduction system and clinical treatment of sinus arrhythmia.

Materials and methods

All study methods were approved by the Ethics Committee of our Medical University.

Materials

Fifteen adult healthy mice of either sex weighing between 25 g and 30 g were used for the study. After the mice were killed by intraperitoneal injection of large-dose pentobarbital sodium (0.3%), their thorax cavities were quickly opened, and then the whole heart was taken. The ventricles were removed from the whole heart at the horizontal position, and the left and right atria and their surrounding connective tissue were retained. The obtained samples were washed with distilled water immediately followed by fixation in 4% paraformaldehyde for 24 h.

Masson trichrome staining

The fixed samples underwent dehydration, and paraffin-embedding at horizontal position, and then were sectioned at a thickness of 5 µm. Samples from 5 mice were used for Masson trichrome staining. Sections were observed under Nikon80i microscope.

NF-160 immunohistochemical staining

The samples from another 5 mice underwent paraffin embedding, and then were sectioned at a thickness of 5 µm. After dewaxed and debenzolization, rabbit-derived NF-160 (Abcam, USA) was added at a dilution of 1:300 at 4°C overnight, and then the sections were washed with TBS at room temperature followed by addition of anti-rabbit horseradish peroxidase at room temperature for one hour. Finally, the sections underwent counterstaining with hematoxylin, dehydration, transparency and mounting with neutral gum.

Immunofluorescence staining

The samples from the remaining 5 mice underwent paraffin embedding, and then were sectioned at a thickness of 5 µm. After dewaxed and debenzolization, rabbit-derived NF-160 with a dilution rate of 1:300 (Abcam, USA) and rat-derived HCN4 with a dilution rate of 1:300 (Abcam, USA) were respectively added at 4°C overnight, and then the sections were washed with TBS at room temperature followed by addition of anti-rabbit FITC with a dilution rate of 1:200 (Boster, Wuhan China) and anti-rat TRITC with a dilution rate of 1:200 (Boster, Wuhan, China), respectively, at room temperature for one hour. Finally, the sections were washed, stained with DAPI and sealed with glycerol followed by observation under a confocal microscope.

Results

Position and morphology of the sinoatrial node

The adult mouse sinoatrial node is located between the sulcus terminalis atrii dextri and superior vena cava. In two mice, the sino-atrial nodal tissue began appearing above the roof of the right atrium. The sinoatrial node was wrapped by connective tissue with a sinus node artery passing though it. Most sinus node cells were not connected with heart atrial muscles and endocardium, only partial cells at the bottom of the sinoatrial node extended to the right atrial endocardium where these cells migrated along with Purkinje fibers in the subendocardial layer. The shape of the mouse sinoatrial node was ovoid or irregular in horizontal sections. The morphology of the sinoatrial node was very different in various horizontal sections.

Cell components of the sinoatrial node

Masson trichrome staining and NF-160 immunohistochemical staining showed plenty of collagen fibers, and cell components including pacemaker cells, transitional cells and interstitial cells. The pacemaker cells were larger than other cells and most of them were located in the central part of the sinoatrial node. The pacemaker cells with obscure boundary had light cytoplasm and large nucleus with nucleoli arranged in clusters or alone. Most of transitional cells were distributed in the periphery of the sinoatrial node. Transitional cells were slightly smaller than pacemaker cells, but the color of cytoplasm and nucleus of transitional cells was darker than that of pacemaker cells. The expression of NF-160 was found in both pacemaker cells and transitional cells, but failed to be found in interstitial cells. The interstitial cells varied in size and scattered throughout pacemaker and transitional cells (Figures 1 and 2).

Figure 1.

Masson trichrome staining of mouse sinoatrial node. A and B: Show the horizontal sections from the central part of the sinoatrial node. The red arrows indicate the sinus node artery passing through the sinoatrial node. C: Shows the junction of sinoatrial node along with Purkinje fibers on the horizontal section from the bottom of the sinoatrial node. The red arrow indicates the sinus node artery. The green arrow indicates Purkinje fibers.

Figure 2.

Immunohistochemical staining of NF-160 in mouse sinoatrial node. (A and B) shows pacemaker cells, transitional cells and connective tissue cells. The green arrows indicate pacemaker cells. The pacemaker cells are large with a big and light nucleus, and are mostly located in the central part of the sinoatrial node. The black arrows indicate transitional cells. The transitional cells with a dark-stained nucleus are smaller than pacemaker cells, and are mostly distributed in the periphery of the sinoatrial node. The connective tissue cells fail to be stained. The expression of NF-160 is found in both pacemaker cells and transitional cells, but fails to be identified in the connective tissue cells. *Indicates sinus node artery. (C and D) shows the bottom of the sinoatrial node which becomes thin and migrates along with Purkinje fibers in the subendocardial layer. The red arrows indicate Purkinje fibers and the black arrow indicates the right atrioventricular valve. Scale bar size: 100 um in (A, C and D); Scale bar size: 50 um in (B).

Immunofluorescence staining of NF-160 and HCN4

Both the peripheral and central parts of the sinoatrial node were covered with NF-160 and HCN4-positive cells, demonstrating that both pacemaker cells and transitional cells all had the expressions of NF-160 and HCN4, namely that both cells all had the distributions of neurofilament and HCN4 channel (Figure 3).

Figure 3.

Immunofluorescence staining of NF-160 and HCN4 in mouse sinoatrial node. A: The horizontal section from the middle upper part of the sinoatrial node; B: The horizontal section from the middle lower part of the sinoatrial node. Both the peripheral and central parts of the sinoatrial node are covered with NF-160 and HCN4-positive cells. ×100.

Discussion

The sinoatrial node is located between the sulcus terminalis atrii dextri and superior vena cava in mammals, but the position of the sinoatrial node is slightly different among various species [6]. In this study, we found that the position of adult mouse sinoatrial node was relatively high between the superior vena cava and the roof of the right atrium, and near the venous sinus. To obtain the integral sinoatrial node, the ventricles were removed from the whole hear at the horizontal position, and the left and right atria and the tissues above and behind the atria were retained. The shape of the mouse sinoatrial node was ovoid or irregular in the horizontal sections. The sinoatrial node was wrapped by connective tissue and was not connected with heart atrial muscles and endocardium, only the part at the bottom of the sinoatrial node became thin and migrated along with Purkinje fibers in the subendocardial layer, which is reported for the first time, and remains to be further confirmed.

The cell components of mouse sinoatrial node are similar to that of other mammals mainly including pacemaker cells, transitional cells and interstitial cells. There were plenty of collagen fibers in the sinoatrial node. The collagen fibers play an important role in maintaining the structure, morphology and cell component of sinoatrial node, in blocking the influence from the surrounding environment and in reducing the conduction velocity within the sinoatrial node [7]. We clearly saw a sinus node artery passing through the sinoatrial node, so the artery can be used as a location marker of the sinoatrial node.

NF-160 is commonly used as a cell marker of cardiac conduction system. In this study, we found NF-160 expression in both pacemaker cells and transitional cells of the sinoatrial node, but there was no NF-160 expression in cardiac muscle cells and connective tissue cells. Our results are consistent with other reports [7-9]. The formation of automatic rhythmicity of the cardiac conduction system is based on pacing ion current. There is a variety of pacing ion currents, but the pacing current is the most important among them. The pacing current, an activated inward ionic current, can restrain excessive hyperpolarzation of pacemaker cells and control spontaneous diastolic depolarization of sinus node cells. The pacing current is regulated by HCN [10]. HCN gene includes 4 subtypes (HCN1, HCN2, HCN3 and HCN4). HCN distributed in pacemaker cells of mouse sinoatrial node mainly belongs to HCN4 [11]. It was reported that mice with HCN4 knockout died before birth, and their embryos showed bradycardia and a reduction of 85% pacing current [12]. Therefore, observation of the distribution of HCN4 in the sinoatrial node is conducive to revealing the relation between the morphology of cardiac conduction system and pacing function. Our results indicated that HCN4 was distributed in both pacemaker cells and transitional cells, demonstrating that both cells are all involved in the electrical activity of sinoatrial node and are associated with sinus pacing.

Disclosure of conflict of interest

None.