不同发育时段大鼠心肌组织中超极化激活环核苷酸门控阳离子通道4阳性细胞的分布特征

doi:10.16098/j.issn.0529-1356.2018.06.010

摘要/Abstract

摘要：

目的探讨超极化激活环核苷酸门控阳离子通道4(HCN4)在不同发育时期大鼠心肌组织中各类细胞的表达规律，为进一步研究HCN4对心肌的作用提供形态学依据。方法出生1 d、1月、3月、6月健康SD大鼠各15只。取新鲜心脏，石蜡切片和冷冻切片，利用免疫组织化学和荧光技术观察不同发育时段大鼠心肌组织HCN4阳性细胞的表达分布情况及形态学特征。利用Western blotting 技术观察左右心室HCN4的蛋白含量。结果免疫组织化学和荧光染色结果显示，HCN4阳性细胞在出生后1 d、1月、3月、6月的血管平滑肌细胞，心内膜和血管的内皮细胞，心外膜的间皮细胞、成纤维细胞，大量心房肌细胞和少量心室肌细胞、传导组织细胞均有表达，但不同部位的表达细胞数量不均；Western blotting结果显示，心室HCN4蛋白随着月龄的增加表达逐渐降低。结论 HCN4在大鼠心肌组织中的表达随年龄的增加逐渐下降，但增殖能力较强的细胞持续存在，提示HCN4可能对心肌组织中的多种细胞的生存起调控作用。

关键词: 心脏, 超极化激活环核苷酸门控阳离子通道4, 免疫组织化学, 免疫荧光, 免疫印迹法, 大鼠

Abstract:

Objective To investigate the changes of hyperpolarization-activated cyclic nucleotide gated cation channels 4(HCN4) expression in the rat heart at different developmental stages and to provide morphological basis for further study of HCN4 on cardiac function. Methods Four groups of rats, 15 rats per group, which were separately born for 1 day, 1 month, 3 months and 6 months were used in this study. The hearts of each group were collected. Paraffin section and frozen section were used to observe the distribution and morphological characteristics of HCN4 positive cells in myocardium of rats at different developmental stages by immunohistochemistry and fluorescence techniques. The protein content of HCN4 in left and right ventricles was observed by Western blotting. Results The immunohistochemical and fluorescence results showed that HCN4 positive cells in 1 day after birth, 1 month, 3 months and 6 months of the vascular smooth muscle cells, endocardial and vascular endothelial cells, epicardial mesothelial cells, fibroblasts, a large number of atrial myocytes, a small amount of cardiomyocytes, and conduction of tissue cells were expressed, but the number of expressed cells was uneven in different parts. Western blotting results showed that the expression of ventricular HCN4 protein decreased with age. Conclusion The expression of HCN4 decreased gradually with the increase of age in the rat myocardium, but the cells with strong proliferative ability are sustained expression. These results suggest that HCN4 may regulate the survival of many cells in myocardium.

Key words: Heart, Hyperpolarization-activated cyclic nucleotide gated cation channels 4, Immunohistochemistry, Immunofluorescence, Western blotting, Rat

张亚南任明芬郭志坤. 不同发育时段大鼠心肌组织中超极化激活环核苷酸门控阳离子通道4阳性细胞的分布特征[J]. 解剖学报, 2018, 49(6): 752-758.

ZHANG Ya-nan REN Ming-fen GUO Zhi-kun. Distribution of hyperpolarization-activated cyclic nucleotide gated cation channels 4 positive cells in rat myocardial tissue at different developmental stages[J]. Acta Anatomica Sinica, 2018, 49(6): 752-758.

参考文献

［1］ Ye B, Nerbonne JM. Proteolytic processing of HCN2 and co-assembly with HCN4 in the generation of cardiac pacemaker channels［J］. J Biol Chem, 2009, 284(38): 25 553-25 559.

［2］ Brioschi C, Micheloni S, Tellez JO, et al. Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node［J］. J Mol Cell Cardiol,2009, 47(2): 221-227.

［3］ Liang X, Wang G, Lin L, et al. HCN4 dynamically marks the first heart field and conduction system precursors［J］. Cir Res, 2013, 113(4): 399-407.

［4］ Verkerk AO, Wilders R, van Borren MM, et al. Pacemaker current (If) in the human sinoatrial node［J］. Eur Heart J, 2007, 28(20):2472-2478.

［5］ Stieber J, Hofmann F, Ludwig A. Pacemaker channels and sinus node arrhythmia ［J］. Trend Cardiovasc Med,2004,14(1) ∶23-28.

［6］ Fern ández-Velasco M, Goren N, Benito G,et al. Regional distribution of hyperpolarization-activated current (If) and hyperpolarization-activated cyclic nucleotide-gated channel mRNA expression in ventricular cells from control and hypertrophied rat hearts ［J］. J Physiol, 2003, 553(2): 395-405.

［7］ Dobrzynski H, Nikolski VP, Sambelashvili AT, et al. Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart ［J］. Cir Res, 2003, 93(11): 1102-1110.

［8］ Marionneau C, Couette B, Liu J, et al. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart ［J］. J physiol, 2005, 562(1): 223-234.

［9］ Chandler NJ,Greener ID,Tellez JO,et al.Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker［J］. Circulation,2009, 119(12): 1562-1575.

［10］ Garcia-Frigola C, Shi Y, Evans SM. Expression of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heart development［J］. Gene Exp Patt, 2003, 3(6): 777-783.

［11］ Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart［J］. Proc Nat Acad of Sci, 2003, 100(25): 15235-15240.

［12］ Zheng YJ. Unnecessity of HCN4 encoded If in pacemaker activity of fetal ventricular cardiomyocyte［D］. Huazhong University of Science and Technology, 2011. (in Chinese)

郑云洁. HCN4 编码的IF在胚胎心室起搏活动中的非必要性［D］. 华中科技大学, 2011.

［13］ Qiao AX, Jing Y, Cai YJ, et al. Expression patterns of hyperpolarization-activated cyclic nucleotide-gated cation 4,connexin 43 and podoplanin in the embryonic mouse heart and development of cardiac conduction system ［J］. Acta Anatomica Sinica, 2012，43(3): 405-411. (in Chinese)

乔爱秀, 景雅, 蔡玉瑾, 等. 超极化激活环核苷酸门控阳离子通道4, 连接蛋白 43 和平足蛋白在小鼠胚胎心的表达与心传导系的发生［J］. 解剖学报, 2012，43(3): 405-411.

［14］ Li CX, Lu X, Guo ZhK. Evidence of mitosis in cardiac myocytes in vitro［J］, Acta Anatomica Sinica，2015,46（1）：63-68. (in Chinese)

李辞霞，卢杏，郭志坤.体外培养状态下心肌细胞有丝分裂的证据，解剖学报，2015, 46 (1):63-68.

［15］ Chang Y, Guo K, Li Q, et al.Multiple directional differentiation difference of neonatal rat fibroblasts from six organs［J］.Cell Physiol Biochem, 2016, 39(1):157-171.

［16］ Bucchi A, Baruscotti M, Robinson RB, et al. Modulation of rate by autonomic agonists in SAN cells involves changes in diastolic depolarization and the pacemaker current［J］. J Mole Cell Cardiol, 2007, 43(1): 39-48.

［17］ Maltsev VA, Lakatta EG. The funny current in the context of the coupled-clock pacemaker cell system［J］,Heart Rhythm,2012,9(2):302-306

［18］ Zicha S, Fernández-Velasco M, Lonardo G, et al. Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model［J］. Cardiovasc Res, 2005, 66(3): 472-481.

［19］ Xia S, Wang Y, Zhang Y, et al. Dynamic changes in HCN2, HCN4, KCNE1, and KCNE2 expression in ventricular cells from acute myocardial infarction rat hearts［J］. Biochem Biophysical Res Communic, 2010, 395(3): 330-335.

［20］ Stillitano F, Lonardo G, Zicha S, et al. Molecular basis of funny current (If) in normal and failing human heart［J］. J Mol Cell Cardiol, 2008, 45(2): 289-299.

［21］ Wang QZh, Bao W, Zhou L, et al. Bone marrow mesenchymal stem cell transplantation effect on expression of HCN2 and HCN4 gene following myocardial infarctio［J］. Chinese Journal of Tissue Engineering Research, 2010, 14(1): 86-90. (in Chinese)

王庆志, 包巍, 周立, 等. 大鼠骨髓间充质干细胞移植梗死心肌 HCN2及HCN4基因的表达［J］. 中国组织工程研究, 2010, 14(1): 86-90.

[1]	洪晓敏李三强崔钦奕郑润月杨孟利罗仁利李前辉 . 酒精性肝纤维化核因子κB信号通路与性别的差异 [J]. 解剖学报, 2024, 55(1): 55-61.
[2]	魏茜茜李超红赵晨露唐家萍刘玉珍. 大鼠颈上神经节中Ⅰ组代谢型谷氨酸受体表达及慢性间歇性低氧对其的影响 [J]. 解剖学报, 2024, 55(1): 3-9.
[3]	袁蕾杨智伟杨惠刘政海何洁万炜. 三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛 [J]. 解剖学报, 2024, 55(1): 25-31.
[4]	马婷婷陈建红刘爱翠李海宁. 抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用 [J]. 解剖学报, 2024, 55(1): 32-42.
[5]	邹成功冯浩陈兵唐辉邵川孙谋杨荣何家全. 创伤性颅脑损伤中神经功能障碍与认知功能损伤的动态变化 [J]. 解剖学报, 2024, 55(1): 43-48.
[6]	吕海燕沈西雅赵富生朱梅. 松茸多糖对 1-甲基-4-苯基-吡啶离子诱导 PC12 细胞损伤的影响 [J]. 解剖学报, 2024, 55(1): 49-54.
[7]	郑丽平刘霞杜晓华吴亚娟刘珊珊 . 脑源性神经营养因子在牦牛与黄牛端脑不同区域的表达与分布 [J]. 解剖学报, 2024, 55(1): 10-16.
[8]	苏芃王兴仪梁景岩熊天庆. 一种低密度及高纯度胎鼠原代海马神经元培养方法的优化及鉴定[J]. 解剖学报, 2024, 55(1): 113-119.
[9]	郝永明郭磊周程辉裴汉军李莉赵哲 . 改良腹主动脉缩窄法建立心肌肥厚模型[J]. 解剖学报, 2024, 55(1): 120-124.
[10]	许亚平余悦欣陈金福王柯柯郭志坤 . 高龄大鼠心室肌间质弹性纤维和胶原纤维的结构分布特征及其机制 [J]. 解剖学报, 2023, 54(6): 716-721.
[11]	刘丽平朱梦妮徐慧 . 白细胞介素6对脂多糖诱导所致类子痫前期大鼠有核红细胞的影响 [J]. 解剖学报, 2023, 54(6): 722-729.
[12]	王文娟丁雨桐苏昊任捷孙艳荣王涵菲陆嘉莉张林倩白钰秦丽华. 低雌激素状态下大鼠海马及纹状体Nogo-A的表达变化[J]. 解剖学报, 2023, 54(6): 620-627.
[13]	张琴杨俊霞蒋青松. 福辛普利对糖尿病视网膜病变小鼠血管紧张素转化酶2和血管内皮生长因子的影响[J]. 解剖学报, 2023, 54(6): 628-634.
[14]	梁盼盼禹爱梅杜静寇文辉王欢欢宋爱霞. 基于c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶信号通路探讨阿魏酸钠对偏头痛大鼠炎症反应的抑制作用[J]. 解剖学报, 2023, 54(6): 652-659.
[15]	徐颖刘斌郑兴何宗钊. 补体C3a受体在盲肠结扎穿孔致脓毒症大鼠认知障碍中的作用及机制 [J]. 解剖学报, 2023, 54(6): 668-675.