Molecular Quiescence and Cardiomyocyte Maturation

分子静止和心肌细胞成熟

• 批准号: 10371079
• 负责人: RICHARD T LEE
• 金额: $ 42.25万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-05 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371079
• 关键词: 3-Dimensional; Adult; Affect; Animal Model; Automobile Driving; Birth; Cardiac Myocytes; Cell Cycle; Cell Cycle Arrest; Cell Therapy; Cells; Coupling; Data; Development; E2F transcription factors; EIF4EBP1 gene; Electrophysiology (science); Embryo; Energy Metabolism; Environment; Eukaryotic Initiation Factor-4E; FRAP1 gene; Family; Fetus; Future; Gene Expression; Gene Expression Profile; Glycolysis; Goals; Growth; Heart failure; Human; Incidence; Laboratories; Lead; Life; Metabolic; Metabolism; Methods; Molecular; Nutrient; Organism; Pathway interactions; Patients; Perinatal; Phenotype; Play; Production; Property; Protocols documentation; Regulation; Role; Signal Pathway; Signal Transduction; Suspension Culture; System; Testing; Time; Translations; Up-Regulation; Ventricular Arrhythmia; base; cardiac tissue engineering; cardiovascular disorder therapy; detection of nutrient; differentiation protocol; fatty acid oxidation; fetal; heart cell; heart function; human stem cells; improved; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; inhibitor; lipid metabolism; overexpression; prevent; protein expression; senescence; stem cells; transcription factor

Stem cell approaches to treat heart failure will require production of mature human cardiomyocytes (CMs) to improve systolic heart function. However, CMs derived from embryonic or induced pluripotent stem cells (iPSCs) remain functionally immature using current approaches. When delivered to adult large animal models, these immature CMs result in potentially life-threatening ventricular arrhythmias. Successful translation of cell therapies for cardiovascular disease will thus likely require defining molecular pathways to mature human stem cell-derived CMs. Cellular quiescence is a temporary non-proliferating state that can last for the lifetime of the organism. Cellular quiescence can be a diverse state with varying depth of quiescence and other molecular conditions that fit within the overall quiescence concept. Based on new preliminary data shown here, we seek to investigate whether cellular quiescence is required for CM maturation from human iPSCs. During development, CMs undergo a shift from a proliferative state as a fetus, to a more mature but quiescent state after birth. This shift is accompanied by a change in energy metabolism, with fetal CMs deriving energy primarily through glycolysis, and adult CMs deriving energy primarily through fatty acid oxidation. The mechanistic target of rapamycin (mTOR) signaling pathway plays a key role in nutrient sensing and growth, and regulation of mTOR affects the metabolic shift from glycolysis to lipid metabolism. Cell cycle arrest with transient mTOR inhibition may lead to cellular quiescence. We hypothesize that regulation of the mTOR pathway is a key driver in CM maturation via driving cells to quiescence. The following Aims will test mechanistic hypotheses to understand how the mTOR signaling pathway and the E2F family of transcription factors can enhance CM maturation and test whether mTOR pathway manipulation in 3D systems also enhances CM maturation. Specific Aim 1: To define the role of 4E-BP1 activation in Torin1-induced maturation of iPSC-derived CMs. We will modulate 4E-BP1 at different stages of CM maturation and determine whether this mechanism explains Torin1-induced CM maturation. We will evaluate electrophysiological properties, contractility, metabolism, and gene and protein expression to characterize CM phenotype and maturation. Specific Aim 2: To define how quiescence depth by E2F affects maturation of iPSC-derived cardiomyocytes. We will perform cell cycle analysis on differentiating or maturing CMs with or without cell cycle inhibitors. We will evaluate whether overexpression of E2F1/2/3a or deletion of E2F3a-8 prevents CM maturation. Specific Aim 3: To explore the role of transient inhibition of mTOR in the maturation of iPSC-derived CMs in a 3D environment. Because mTOR signaling can differ in 2D versus 3D environments, we seek to test whether mTOR inhibition increases contractility and enhances excitation-contraction coupling in CMs in 3D.

干细胞治疗心力衰竭将需要产生成熟的人类心肌细胞 (CMS)改善收缩心功能。然而，CMS来源于胚胎或诱导的多能干细胞 使用目前的方法，细胞(IPSCs)在功能上仍然不成熟。当分娩给成年的大型动物时 这些未成熟的CMS会导致潜在的危及生命的室性心律失常。成功 因此，心血管疾病的细胞疗法的翻译可能需要定义分子途径来 成熟的人类干细胞来源的CMS。细胞静止是一种暂时的非增殖状态，可以持续 在有机体的整个生命周期内。细胞静止可以是不同的状态，具有不同的静止深度 以及其他符合整体静止概念的分子条件。根据新的初步数据 如图所示，我们试图研究人类CM成熟是否需要细胞静止 IPSCs。在发育过程中，CMS经历了从胎儿时期的增殖状态到更成熟但 出生后处于静止状态。这种转变伴随着能量代谢的变化，胎儿CMS 主要通过糖酵解获得能量，成年CMS主要通过脂肪酸获得能量 氧化。雷帕霉素(MTOR)信号通路的机制靶点在营养感知中起着关键作用 和生长，mTOR的调节影响代谢从糖酵解到脂代谢的转变。细胞周期 短暂的mTOR抑制引起的细胞停滞可能导致细胞静止。我们假设，对 MTOR途径通过驱动细胞静止是CM成熟的关键驱动因素。以下目标将考验 理解mTOR信号通路和E2F转录家族的机械论假说 因子可以促进CM成熟，并测试在3D系统中是否也有mTOR通路操作 促进CM成熟。 特异性目标1：确定4E-BP1激活在Torin1诱导IPSC来源的成熟中的作用 不育系CMS。我们将在CM成熟的不同阶段调节4E-BP1，并确定这一机制是否 解释了Torin1诱导的CM成熟。我们将评估电生理特性，收缩性能， 代谢，基因和蛋白的表达，以表征CM的表型和成熟。 特定目标2：确定E2F的静止深度如何影响IPSC来源的成熟 心肌细胞。我们将对分化或成熟的CMS进行细胞周期分析，无论有没有细胞周期 抑制剂。我们将评估E2F1/2/3a的过度表达或E2F3a-8的缺失是否会阻止CM 成熟。 特异性目标3：探讨mTOR的瞬时抑制在IPSC来源的成熟中的作用 3D环境中的CMS。由于mTOR信令在2D和3D环境中可能不同，我们寻求测试 MTOR抑制是否增加CMS的收缩能力和增强兴奋-收缩偶联。