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Molecular Quiescence and Cardiomyocyte Maturation

分子静止和心肌细胞成熟

基本信息

批准号：
10589890
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/10589890
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 Life Metabolic Metabolism Methods Molecular Nutrient availability 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 cardiac tissue engineering cardiovascular disorder therapy detection of nutrient differentiation protocol efficacy evaluation 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。