Stem Cell & Regenerative Medicine Program, Research Centre of Heart, Brain, Hormone and Healthy Ageing, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China.
J Mol Cell Cardiol. 2010 Jun;48(6):1129-37. doi: 10.1016/j.yjmcc.2010.01.015. Epub 2010 Jan 29.
Hypoxia plays an important role in the proliferation, differentiation and maintenance of the cardiovascular system during development. While low oxygen tension appears to direct the cultured embryonic stem cells (ESCs) to differentiate into cardiomyocytes, the underlying molecular mechanism remains unclear. At a molecular level, hypoxia inducible factor-1 (HIF-1) plays an important role in handling the hypoxia signal. In the present study, we demonstrated that expression of exogenous HIF-1 alpha cDNA into murine ESCs significantly promoted cardiogenesis as indicated by a higher percentage of beating embryoid body and troponin-T positive cell counts as well as increased expression of early and late cardiac markers, such as GATA-binding protein 4 and 6, NK2 transcription factor related locus 5, alpha-myosin heavy chain, beta-myosin heavy chain and myosin light chain 2 ventricular transcripts. In addition, the transduced cells exhibited increased mRNA levels of cardiotrophin-1 and vascular endothelial growth factor, along with phosphorylation of eNOS [p-eNOS (ser1171)]. Application of NOS inhibitors, diphenyleneiodonium chloride (DPI), N(omega)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) or N(omega)-Nitro-L-arginine (L-NNA) abolished the HIF-1 alpha stimulated cardiac differentiation. With the clues of upregulated mRNA expression of calcium handling proteins, ryanodine receptor 2, sodium calcium exchanger and sarcoplasmic/endoplasmic reticulum calcium ATPase, in the transduced HIF-1 alpha ESCs, further study indicated that the maximum upstroke and decay velocity was significantly increased in both non-caffeine and caffeine-induced calcium transient in ESCs-derived cardiomyocytes. This suggests a well developed function of the sarcoplasmic reticulum in ESC-derived cardiomyocytes. Electrophysiological study also indicated that a portion of the HIF-1 alpha-transduced cells exhibited prominent phase-4 depolarization. These findings suggest that keen activation of the HIF-1 pathway enhances differentiation and maturation of cardiomyocytes derived from ESCs.
缺氧在胚胎心血管系统的增殖、分化和维持中起着重要作用。虽然低氧张力似乎指导培养的胚胎干细胞（ESCs）分化为心肌细胞，但潜在的分子机制尚不清楚。在分子水平上，缺氧诱导因子-1（HIF-1）在处理缺氧信号中起着重要作用。在本研究中，我们证明了外源性 HIF-1α cDNA 的表达显著促进了鼠 ESCs 的心肌发生，表现为搏动胚体的比例更高，肌钙蛋白-T 阳性细胞计数增加，以及早期和晚期心脏标志物的表达增加，如 GATA 结合蛋白 4 和 6、NK2 转录因子相关基因座 5、α-肌球蛋白重链、β-肌球蛋白重链和肌球蛋白轻链 2 心室转录物。此外，转导细胞表现出心肌营养素-1 和血管内皮生长因子的 mRNA 水平升高，以及内皮型一氧化氮合酶（eNOS）[p-eNOS（ser1171）]的磷酸化。NOS 抑制剂二苯基碘（DPI）、N（ω）-硝基-L-精氨酸甲酯盐酸盐（L-NAME）或 N（ω）-硝基-L-精氨酸（L-NNA）的应用消除了 HIF-1α 刺激的心脏分化。根据钙处理蛋白、兰尼碱受体 2、钠钙交换蛋白和肌浆网/内质网钙 ATP 酶的 mRNA 表达上调的线索，在转导的 HIF-1α ESCs 中，进一步的研究表明，在 ESCs 衍生的心肌细胞中，非咖啡因和咖啡因诱导的钙瞬变中的最大上升和下降速度显著增加。这表明 ESCs 衍生的心肌细胞中肌浆网功能发育良好。电生理研究还表明，一部分 HIF-1α 转导细胞表现出明显的相 4 去极化。这些发现表明，HIF-1 途径的强烈激活增强了 ESCs 衍生的心肌细胞的分化和成熟。
