Liu Jiaying, Kasuya Go, Zempo Buntaro, Nakajo Koichi
Division of Integrative Physiology, Department of Physiology, Jichi Medical University, Shimotsuke, Japan.
Front Physiol. 2022 Jun 30;13:901571. doi: 10.3389/fphys.2022.901571. eCollection 2022.
The HCN4 channel is essential for heart rate regulation in vertebrates by generating pacemaker potentials in the sinoatrial node. HCN4 channel abnormality may cause bradycardia and sick sinus syndrome, making it an important target for clinical research and drug discovery. The zebrafish is a popular animal model for cardiovascular research. They are potentially suitable for studying inherited heart diseases, including cardiac arrhythmia. However, it has not been determined how similar the ion channels that underlie cardiac automaticity are in zebrafish and humans. In the case of HCN4, humans have one gene, whereas zebrafish have two ortholog genes ( and ; 'Dr' referring to ). However, it is not known whether the two HCN4 channels have different physiological functions and roles in heart rate regulation. In this study, we characterized the biophysical properties of the two zebrafish HCN4 channels in oocytes and compared them to those of the human HCN4 channel. We found that they showed different gating properties: DrHCN4L currents showed faster activation kinetics and a more positively shifted G-V curve than did DrHCN4 and human HCN4 currents. We made chimeric channels of DrHCN4 and DrHCN4L and found that cytoplasmic domains were determinants for the faster activation and the positively shifted G-V relationship in DrHCN4L. The use of a dominant-negative HCN4 mutant confirmed that DrHCN4 and DrHCN4L can form a heteromultimeric channel in oocytes. Next, we confirmed that both are sensitive to common HCN channel inhibitors/blockers including Cs, ivabradine, and ZD7288. These HCN inhibitors successfully lowered zebrafish heart rate during early embryonic stages. Finally, we knocked down the HCN4 genes using antisense morpholino and found that knocking down either or both of the HCN4 channels caused a temporal decrease in heart rate and tended to cause pericardial edema. These findings suggest that both DrHCN4 and DrHCN4L play a significant role in zebrafish heart rate regulation.
HCN4通道通过在窦房结产生起搏电位，对脊椎动物的心率调节至关重要。HCN4通道异常可能导致心动过缓和病态窦房结综合征，使其成为临床研究和药物研发的重要靶点。斑马鱼是心血管研究中常用的动物模型。它们有可能适用于研究包括心律失常在内的遗传性心脏病。然而，尚未确定斑马鱼和人类中构成心脏自律性基础的离子通道有多相似。就HCN4而言，人类有一个基因，而斑马鱼有两个直系同源基因（和；“Dr”指）。然而，尚不清楚这两个HCN4通道在心率调节中是否具有不同的生理功能和作用。在本研究中，我们在卵母细胞中表征了两种斑马鱼HCN4通道的生物物理特性，并将它们与人类HCN4通道的特性进行了比较。我们发现它们表现出不同的门控特性：与DrHCN4和人类HCN4电流相比，DrHCN4L电流表现出更快的激活动力学和更正向偏移的G-V曲线。我们构建了DrHCN4和DrHCN4L的嵌合通道，发现细胞质结构域是DrHCN4L中更快激活和正向偏移的G-V关系的决定因素。使用显性负性HCN4突变体证实，DrHCN4和DrHCN4L可以在卵母细胞中形成异源多聚体通道。接下来，我们证实两者都对包括Cs、伊伐布雷定和ZD7288在内的常见HCN通道抑制剂/阻滞剂敏感。这些HCN抑制剂在胚胎早期成功降低了斑马鱼的心率。最后，我们使用反义吗啉代敲低HCN4基因，发现敲低HCN4通道中的一个或两个都会导致心率暂时下降，并倾向于导致心包水肿。这些发现表明，DrHCN4和DrHCN4L在斑马鱼心率调节中都发挥着重要作用。
