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.

治疗心力衰竭的干细胞方法需要产生成熟的人类心肌细胞（CM）改善心脏收缩功能。然而，源自胚胎或多能干细胞的 CM 使用目前的方法，细胞（iPSC）在功能上仍然不成熟。当交付给成年大型动物时在模型中，这些不成熟的 CM 会导致潜在危及生命的室性心律失常。成功的因此，心血管疾病细胞疗法的转化可能需要定义分子途径成熟的人类干细胞衍生的 CM。细胞静止是一种可以持续的暂时的非增殖状态对于有机体的一生。细胞静止可以是具有不同静止深度的多种状态以及符合整体静止概念的其他分子条件。基于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 iPSC。在发育过程中，CM 经历了从胎儿的增殖状态转变为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 CM 主要通过糖酵解获取能量，成人 CM 主要通过脂肪酸获取能量氧化。雷帕霉素（mTOR）信号通路的机制靶标在营养传感中发挥关键作用 mTOR 的生长和调节影响从糖酵解到脂质代谢的代谢转变。细胞周期短暂的 mTOR 抑制可能导致细胞静止。我们假设监管 mTOR 通路通过驱动细胞进入静止状态，是 CM 成熟的关键驱动因素。将测试以下目标机制假设，以了解 mTOR 信号通路和 E2F 家族的转录过程因素可以促进 CM 成熟并测试 3D 系统中的 mTOR 通路操纵是否也有效促进 CM 成熟。具体目标 1：确定 4E-BP1 激活在 Torin1 诱导的 iPSC 衍生成熟中的作用 CM。我们将在CM成熟的不同阶段调节4E-BP1并确定该机制是否有效解释了 Torin1 诱导的 CM 成熟。我们将评估电生理特性、收缩性、代谢、基因和蛋白质表达来表征 CM 表型和成熟。具体目标 2：确定 E2F 的静止深度如何影响 iPSC 衍生的成熟心肌细胞。我们将对具有或不具有细胞周期的分化或成熟的 CM 进行细胞周期分析抑制剂。我们将评估 E2F1/2/3a 的过度表达或 E2F3a-8 的缺失是否可以预防 CM 成熟。具体目标 3：探讨 mTOR 瞬时抑制在 iPSC 衍生细胞成熟中的作用 3D 环境中的 CM。由于 mTOR 信号传导在 2D 与 3D 环境中可能有所不同，因此我们寻求测试 mTOR 抑制是否会增加收缩性并增强 3D CM 中的兴奋-收缩耦合。