Cardiology Division, Department of Medicine, Queen Mary Hospital, University of Hong Kong and Research Center of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China.
Acta Pharmacol Sin. 2011 Jan;32(1):52-61. doi: 10.1038/aps.2010.188. Epub 2010 Dec 13.
To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells (mESCs).
Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain (20 μmol/L) for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging.
Compared with control, mESCs treated with ouabain (20 μmol/L) yielded a significantly higher percentage of cardiomyocytes, and significantly increased expression of a panel of cardiac markers including Nkx 2.5, α-MHC, and β-MHC. The α1 and 2- isoforms Na(+)/K(+)-ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erk1/2 pathway by U0126 (10 μmol/L) inhibited cardiac differentiation while ouabain (20 μmol/L) rescued the effect. Interestingly, the expression of calcium handling proteins, including ryanodine receptor (RyR2) and sacroplasmic recticulum Ca(2+) ATPase (SERCA2a) was also upregulated in ouabain-treated mESCs. ESC-derived cardiomyocyes (CM) treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca(2+) imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity (P<0.01) and maximum decay velocity (P<0.05), as well as a higher amplitude of caffeine induced Ca(2+) transient (P<0.05), suggesting more mature sarcoplasmic reticulum (SR).
Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.
研究强心甾苷哇巴因对鼠胚胎干细胞（mESCs）心脏分化的影响。
采用标准悬滴法，在 20μmol/L 哇巴因存在的条件下增强鼠胚胎干细胞的心脏分化 7d。通过流式细胞术、RT-PCR 和共聚焦钙成像检测分离的 ES 衍生心肌细胞。
与对照组相比，哇巴因（20μmol/L）处理的 mESCs 产生的心肌细胞比例显著增加，心肌标志物包括 Nkx2.5、α-MHC 和β-MHC 的表达显著增加。分化过程中 mESCs 中也增加了哇巴因作用的钠/钾-ATP 酶的α1和2-同工型。在心脏肥大途径中涉及的三种 MAPK 中，哇巴因增强了 ERK1/2 的激活。U0126（10μmol/L）阻断 Erk1/2 通路抑制心脏分化，而哇巴因（20μmol/L）则挽救了这种作用。有趣的是，钙处理蛋白的表达，包括肌浆网钙释放通道（RyR2）和肌浆网 Ca2+-ATP 酶（SERCA2a）也在哇巴因处理的 mESCs 中上调。哇巴因处理的 ESC 衍生心肌细胞（CM）似乎具有更成熟的钙处理能力。如共聚焦 Ca2+成像所示，哇巴因处理的 mESCs 分离的心肌细胞表现出更高的最大上升速度（P<0.01）和最大衰减速度（P<0.05），以及更高的咖啡因诱导的 Ca2+瞬变幅度（P<0.05），提示更成熟的肌浆网（SR）。
哇巴因通过激活 Erk1/2 和更成熟的 SR 处理钙来诱导 mESC 衍生心肌细胞的心脏分化和成熟。
