Molecular Quiescence and Cardiomyocyte Maturation

分子静止和心肌细胞成熟

基本信息

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
项目类别：
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

项目摘要

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的方法需要产生成熟的人类心肌细胞（CMS）改善收缩性心脏功能。但是，源自胚胎或诱导多能茎的CM 使用当前方法，细胞（IPSC）在功能上保持不成熟。当送到成年大型动物时模型，这些未成熟的CM会导致潜在的威胁生命的心律不齐。成功的因此，对心血管疾病的细胞疗法的翻译可能需要定义分子途径成熟的人类干细胞衍生的CM。细胞静止是一种暂时的非增殖状态，可以持续在生物体的一生中。细胞静止可以是多种状态，静止深度变化以及适合整体静止概念的其他分子条件。基于新的初步数据这里显示的是，我们试图研究人类CM成熟是否需要细胞静止 ipscs。在开发过程中，CMS经历了从胎儿的增殖状态转变为更成熟但出生后静止状态。这种转变伴随着能量代谢的变化，胎儿CMS 主要通过糖酵解和成年CMS得出能量，主要通过脂肪酸得出能量氧化。雷帕霉素（MTOR）信号通路的机械靶标在营养感应中起关键作用和生长以及MTOR的调节会影响从糖酵解到脂质代谢的代谢转移。细胞周期瞬时抑制作用可能导致细胞静止。我们假设对 MTOR途径是CM成熟的关键驱动器，通过驱动细胞静止。以下目标将测试机械假设，以了解MTOR信号通路和E2F转录家族如何因素可以增强CM成熟并测试3D系统中MTOR途径是否操纵增强CM成熟。特定目标1：定义4E-BP1激活在TORIN1诱导的IPSC衍生的成熟中的作用 CMS。我们将在CM成熟的不同阶段调节4E-BP1，并确定该机制是否解释了Torin1诱导的CM成熟。我们将评估电生理特性，收缩力，代谢，基因和蛋白质表达以表征CM表型和成熟。特定目的2：定义E2F的静止深度如何影响IPSC衍生的成熟心肌细胞。我们将在有或没有细胞周期的情况下对分化或成熟CMS进行细胞周期分析抑制剂。我们将评估E2F1/2/3A的过表达或E2F3A-8的删除是否可以防止CM 成熟。特定目的3：探索MTOR瞬时抑制在IPSC衍生的成熟中的作用 CMS在3D环境中。由于MTOR信令在2D与3D环境中可能有所不同，因此我们试图测试 MTOR抑制是否会增加收缩性并增强3D中CMS中的激发反应耦合。