Ascorbic acid-mediated enhanced cardiomyocyte differentiation of mouse ES-cells involves interplay of DNA methylation and multiple-signals
Ascorbic acid-mediated enhanced cardiomyocyte differentiation of mouse ES-cells involves interplay of DNA methylation and multiple-signals
复制标题
DOI：
10.1016/j.diff.2017.04.001
复制
发表时间：
2017-07-01
期刊：
影响因子：
2.9
通讯作者：
Seshagiri, Polani B.
中科院分区：
文献类型：
--
作者：
Abbey, Deepti;Seshagiri, Polani B.
胚胎干细胞（ES 细胞）为研究谱系特异性分化提供了良好的模型系统。尽管 ES 细胞向心肌细胞的分化已有记录，但尚未实现对分化分子机制的清晰理解和提高功能分化效率。在这方面，抗坏血酸 (Aa) 被证明是 ES 细胞中有效的心肌诱导剂之一。但是，人们对其机制知之甚少。因此，我们研究了 Aa 介导的 ES 细胞心肌细胞分化的机制。在这里，我们描述了表观遗传（DNA 甲基化）以及整合素和 Erk 信号系统在心肌细胞分化过程中的潜在参与。转基因 GS-2 ES 细胞和野生型 D3 ES 细胞在存在或不存在 Aa 以及存在或不存在 Erk、胶原蛋白和整联蛋白途径抑制剂的情况下分化为心肌细胞。在特定时间点，通过基因表达分析和功能性cTnI(+)心肌细胞的比例对ES细胞的分化状态进行评分。使用甲基化特异性 PCR (MSP) 分析 Aa 刺激后心源性细胞中 Isl-1、BMP-2、GATA-4 和 α-MHC 的 DNA 甲基化变化。我们观察到，当在 ES 细胞分化的初始阶段应用 Aa 时，与自发分化期间观察到的情况 (70%) 相比，Aa 持续增强了心脏分化 (99%)。这与心脏发生相关基因的表达增强有关。观察到 cTnI(+) 细胞增加两倍，肌原纤维排列适当。观察到的 Aa 效应是由于胶原蛋白和整合素信号传导增强，加上下游 p-ERK1/2 的高表达。此外，还观察到 DNA 甲基化参与调节心脏基因 Isl-1 和 a-MHC 的表达。总体而言，这项研究首次证明，Aa 介导的心脏增强是通过心脏基因（Isl-1 和 a-MHC）DNA 甲基化的表观遗传变化与整合素信号系统的相互作用实现的。
Embryonic stem cells (ES-cells) provide a good model system to study lineage-specific differentiation. Though, the differentiation of ES-cells to cardiomyocytes is documented, a clear understanding of the molecular mechanism of differentiation and improved functional-differentiation efficiency are yet to be achieved. In this regard, ascorbic acid (Aa) is shown to be one of the effective cardiac inducers in ES-cells. But, its mechanism is poorly understood. We therefore, investigated the mechanism of Aa-mediated cardiomyocyte differentiation of ES-cells. Here, we describe the potential involvement of epigenetic (DNA methylation) as well as integrin-and Erk-signaling systems during cardiomyocyte differentiation. Transgenic GS-2 ES-cells and wild-type D3 ES-cells were differentiated to cardiomyocytes, in the presence or absence of Aa and with or without inhibitors of Erk-, collagen-and integrin-pathways. At specific time points, differentiated states of ES-cells were scored by gene expression analyses and the proportion of functional cTnI(+) cardiomyocytes. DNA methylation changes of Isl-1, BMP-2, GATA-4 and alpha-MHC in cardiogenic cells, following stimulation with Aa, were analyzed by using methylation specific PCR (MSP). We observed that Aa, when applied in initial phase of ES-cell differentiation, consistently enhanced cardiac differentiation (99%) over that observed during spontaneous differentiation (70%). This was associated with enhanced expressions of cardiogenesis-associated genes. A two-fold increase in cTnI(+) cells was observed, with appropriate myofibril arrangement. The observed effect of Aa was due to enhanced collagen and integrin signaling, coupled with a high p-ERK1/2 expression, downstream. Besides, the involvement of DNA methylation in regulating the expression of cardiac genes i.e., Isl-1 and a-MHC was also observed. Overall, this study, for the first time, demonstrates that Aa-mediated cardiac enhancement is brought about, mechanistically, through the interplay of epigenetic changes in DNA methylation of cardiac genes (Isl-1 and a-MHC) and integrin signaling system.