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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.

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 (CMs)改善心脏收缩功能然而，来自胚胎或诱导多能干细胞的CM 使用目前的方法，多能干细胞（iPSC）在功能上仍然不成熟。当交付给成年大型动物时这些不成熟的CM导致潜在的危及生命的室性心律失常。成功因此，将细胞疗法转化为心血管疾病可能需要定义分子途径，成熟人干细胞衍生的CM。细胞静止是一种暂时的非增殖状态， for the lifetime寿命of the organism有机体.细胞静止可以是具有不同静止深度的不同状态以及其他符合整体静止概念的分子条件。根据新的初步数据在这里，我们试图研究细胞静止是否需要从人类CM成熟 iPSCs。在发育过程中，CM经历了从胎儿时的增殖状态到更成熟但出生后的静止状态这种转变伴随着能量代谢的变化，主要通过糖酵解获得能量，成年CM主要通过脂肪酸获得能量氧化雷帕霉素靶蛋白（mTOR）信号通路在营养传感中起着关键作用和生长，并且mTOR的调节影响从糖酵解到脂质代谢的代谢转变。细胞周期瞬时mTOR抑制的停滞可能导致细胞静止。我们假设， mTOR途径是CM成熟的关键驱动因素，通过驱动细胞静止。以下目标将测试理解mTOR信号通路和E2 F家族转录的机制假说因子可以增强CM成熟，并测试3D系统中的mTOR通路操纵是否也促进CM成熟。具体目的1：确定4 E-BP 1活化在Torin 1诱导的iPSC衍生的细胞成熟中的作用。 CM我们将在CM成熟的不同阶段调节4 E-BP 1，并确定这种机制是否解释了Torin 1诱导的CM成熟。我们将评估电生理特性，收缩性，代谢以及基因和蛋白质表达来表征CM表型和成熟。具体目标2：定义E2 F的静止深度如何影响iPSC衍生的细胞的成熟。心肌细胞我们将对分化或成熟的CM进行细胞周期分析，无论是否有细胞周期抑制剂的我们将评估E2 F1/2/3a的过表达或E2 F3 a-8的缺失是否可以预防CM 成熟具体目的3：探索瞬时抑制mTOR在iPSC衍生的细胞成熟中的作用。 3D环境中的CM。由于mTOR信号在2D与3D环境中可能不同，我们试图测试 mTOR抑制是否增加收缩性并增强3D中CM的兴奋-收缩偶联。