Mechanism by which endothelin-1 regulates the differentiation of P19 cells into cardiac conduction cells

doi:10.3969/j.issn.2095-4344.0812

Abstract

Abstract:

BACKGROUND: Endothelin-1, as a paracrine cytokine derived from endocardium and vascular endothelial cells, can promote the differentiation and maturation of cardiac conduction cells.
OBJECTIVE: To explore the effect of endothelin-1 on P19 cells differentiating into cardiac conduction cells in vitro.
METHODS: P19 cells were cultured with 1% dimethyl sulfoxide or 100 nmol/L endothelin-1. Afterwards, the changes of early cardiomyocyte transcription factors MEF2C and GATA4, differentiation markers MHC-α and cTnT, important conduction cell transcription factors Nkx2.5 and Tbx5, and differentiation markers Cx40 and ANP were identified by western blot assay, immunofluorescence and real-time PCR. Specific antagonists of endothelin-1 receptors, and blocking PI3K-AKT-mTOR signaling pathway on the differentiation of P19 cells induced by endothelin-1 were evaluated by real-time PCR.
RESULTS AND CONCLUSION: Endothelin-1 significantly up-regulated the levels of early cardiac transcription factors, cardiomyocyte structure factors and characteristic conduction system markers. Compared with dimethyl sulfoxide, endothelin-1 was prone to enhance the differentiation of cardiac conduction cells derived from P19 cells. Up-regulation of Nkx2.5 and Cx40 by endothelin-1 was mainly attributed to the ETA signaling pathway. ETB signaling pathway may be also involved in a negative feedback regulation pathway. The PI3K-AKT-mTOR signaling pathway can play an important role in the differentiation from P19 cells to cardiac conduction cells triggered by endothelin-1.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Embryonal Carcinoma Stem Cells, Heart Conduction System, Cell Differentiation, Tissue Engineering

CLC Number:

R318

Chen Hao1, Zhang Wu2, Wang Hao1, Zhang Heng-yi1, Xu Zhi-wei1. Mechanism by which endothelin-1 regulates the differentiation of P19 cells into cardiac conduction cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(12): 1915-1921.

Figures/Tables 2

2.1 P19细胞分化过程的形态学变化正常体外培养的P19细胞胞体小，胞质较多，外形呈椭圆或正多边形，边缘有时有细小突起。将P19细胞移入细菌培养皿悬浮培养，同时加入1%二甲基亚砜或内皮素1诱导分化后，P19细胞开始聚集成类似体内拟胚体结构的细胞团继续生长(图1A，B)。诱导4 d后，将P19细胞聚集体移入细胞培养皿中，以无二甲基亚砜和内皮素1的普通生长培养液培养。24 h内多数细胞重新贴壁生长，细胞聚集体由球形变为扁平状多细胞层结构(图1C)。部分发生分化的P19细胞胞核变大，外形由椭圆或正多边形转变为长梭形或纺锤形。 2.2 内皮素1诱导P19细胞分化的浓度选择检测了不同浓度内皮素1对早期心肌分化特征性基因Nkx2.5和MEF2C表达的影响。与对照组相比，10-50 nmol/L的内皮素1对Nkx2.5表达没有明显影响，浓度上升为100 nmol/L时Nkx2.5表达增加明显，200 nmol/L时Nkx2.5表达与其余各组相比均有显著差异(图2A)。另一方面，当内皮素1浓度由10 nmol/L上升为100 nmol/L时，MEF2C表达亦相应增加，100 nmol/L浓度时MEF2C表达与其余各组差异明显；但浓度增加至200 nmol/L时，MEF2C表达反而降低(图2B)。因此，实验选择100 nmol/L内皮素1进行诱导分化实验。 2.3 内皮素1和二甲基亚砜诱导P19细胞向早期心肌细胞分化除Nkx2.5和MEF2C，还选取了早期心肌分化的另一个特征性基因GATA-4，运用Real-time PCR对二甲基亚砜和内皮素1诱导P19细胞分化过程中以上基因的表达进行了检测。结果发现，二甲基亚砜和内皮素1组Nkx2.5表达水平在第8-10天达到峰值后逐渐减低；双因素重复测量方差分析显示，内皮素1组Nkx2.5表达水平与对照组和二甲基亚砜组相比均有明显差异(图3A)。虽然内皮素1组早期GATA-4表达水平有所降低；但总体而言，两组细胞GATA-4表达水平均呈逐渐升高趋势，后期表达水平均显著高于对照组。此外，10-14 d期间内皮素1组GATA-4表达也明显超过二甲基亚砜组(图3B)。作为早期心肌特征性转录因子，MEF2C对调控心室肌分化有重要作用。结果显示，两组细胞第8天起MEF2C表达水平逐渐增加并在第12天达峰；同样，内皮素1组的峰值水平要显著高于二甲基亚砜组和对照组(图3C)。结果说明，二甲基亚砜和内皮素1均能显著上调P19细胞早期心肌分化相关基因的表达水平。Nkx2.5基因升高出现时间最早，达峰时间也最早，在第8天左右；GATA-4和MEF2C基因的表达时间稍晚于Nkx2.5，达峰时间在第12天左右。 2.4 内皮素1能够进一步诱导P19细胞向心肌传导细胞分化随后检测了代表成熟工作心肌的肌节结构基因MHC-α的表达情况。与上述结果不同，虽然第8天起两组细胞MHC-α表达水平均开始增加并在第12天达峰，但内皮素1组表达水平明显不及二甲基亚砜组；并且与对照组相比，内皮素1组的表达水平并无显著提高(图3D)。相反，与对照组和二甲基亚砜组相比，内皮素1则能够显著上调P19细胞体内作为心脏传导细胞特征基因Cx40的表达水平，呈明显升高趋势(图3E)。"

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