Miake Junichiro, Notsu Tomomi, Higaki Katsumi, Hidaka Kyoko, Morisaki Takayuki, Yamamoto Kazuhiro, Hisatome Ichiro
Department of Molecular Medicine and Therapeutics, Faculty of Medicine, Tottori University, Yonago, Japan.
Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago, Japan.
PLoS One. 2017 Aug 17;12(8):e0183225. doi: 10.1371/journal.pone.0183225. eCollection 2017.
Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2'-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration.
心脏祖细胞的增殖能力有限。含富含Glu/Asp的羧基末端结构域的CREB结合蛋白/p300相互作用反式激活因子（Cited）基因家族可调节基因转录。成年小鼠中Cited4基因表达增加与运动诱导的心肌细胞肥大和增殖有关。然而，Cited4基因在心脏发生过程中的表达模式和功能作用在很大程度上尚不清楚。因此，在本研究中，我们调查了Cited4基因在体外心脏发生过程中的表达模式和功能作用。利用小鼠胚胎干细胞形成的胚状体，我们通过定量逆转录-聚合酶链反应评估了Cited4基因的表达模式。Cited4基因表达水平在心脏发生的早期和晚期分别升高和降低。此外，Cited4基因水平在心脏祖细胞群体中显著较高。使用流式细胞术对心脏祖细胞中的Cited4基因进行功能分析表明，与对照组和敲低组相比，Cited4基因的过表达显著增加了心脏祖细胞群体。一项细胞增殖分析，在心脏发生后期检测5-乙炔基-2'-脱氧尿苷掺入和Ki67表达，结果表明，过表达组中具有增殖能力的肌钙蛋白T阳性胚胎干细胞来源的心肌细胞数量显著多于对照组和敲低组。我们的研究结果表明，Cited4基因与体外心脏发生过程中胚胎干细胞来源的心肌细胞的心脏分化和增殖能力的维持有关。因此，操纵Cited4基因表达可能对心脏再生具有重要意义。
