Engineered Stem Cells for Cardiac Repair

用于心脏修复的工程干细胞

基本信息

批准号：
10588153
负责人：
MICHAEL REGNIER
金额：
$ 78.83万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10588153
关键词：
ATP Synthesis Pathway ATP phosphohydrolase Actins Affect Award Behavior Binding Binding Sites Canis familiaris Cardiac Cardiac Myocytes Cardiac Myosins Cell Line Cell Survival Cell Transplantation Cells Cellular Metabolic Process Chronic Computer Models Congestive Heart Failure Contractile Proteins Contracts Data Dependence Depressed mood Development Disease Dose Electrostatics Engineering Engraftment Enzymes Equilibrium Family suidae Funding Gap Junctions Genes Genetic Transcription Goals Heart Heart failure Human Human Engineering In Vitro Infarction Ischemia Kinetics Left Left Ventricular Function Macaca nemestrina Mechanics Mediating Microscopy Modeling Molecular Movement Disorders Mus Muscle Muscle Cells Muscle Contraction Muscle relaxation phase Myocardial Myocardial Infarction Myocardium Myosin ATPase Nucleotides Nude Rats Pathology Pathway interactions Performance Perfusion Phosphocreatine Phosphorylation Physiologic intraventricular pressure Positioning Attribute Proteins Publishing Rattus Recovery Relaxation Reperfusion Injury Reporting Rest Ribonucleotide Reductase Rodent Rodent Model Roentgen Rays Sarcomeres Stimulant Structure Testing Thick Filament Thin Filament Tissues Transgenic Mice Transgenic Organisms Transplantation Ventricular Ventricular Function Vertebral column Viral Vector Work X ray diffraction analysis cardiac repair cell replacement therapy engineered stem cells experimental study functional improvement heart function human stem cells imaging approach improved improved outcome induced pluripotent stem cell inhibitor molecular dynamics multi-scale modeling nonhuman primate novel overexpression pressure promoter recruit single molecule small molecule small molecule therapeutics stem cells targeted treatment time use ultra high resolution

项目摘要

ABSTRACT. Aim 1 of this project is built around years of collaborative work between Drs. Regnier and Murry studying human stem cell derived cardiomyocytes as a potential cell replacement strategy for cardiac repair following myocardial infarction (MI). We have shown that human stem cells can be differentiated into cardiomyocytes (CMs), produced at a scale and purity that permit testing in rodent models and non-human primates (NHP; Macaca nemestrina) and that these cells engraft and integrate with host tissue to improve left ventricular performance. The premise for the proposed experiments is based on two fundamental discoveries: 1) 2-deoxy ATP (dATP) is a potent natural nucleotide stimulant of contractility when used by cardiac myosin, and 2) hiPSC-CMs that overexpress the rate-limiting enzyme for dATP synthesis, ribonucleotide reductase (RNR), have increased contractility and also deliver dATP to the native myocardium heart via gap junctions. In our current award we made excellent progress in testing the hypothesis that engineered hiPSC-CMs with elevated RNR (hiPSC-CMRNR) improve outcomes in cell replacement therapy for MI (compared with control hiPSC-CMs). For this proposal, we have generated new hiPSC-CM lines with gene-edited RNR and different transcriptional promotors. These cells have greater RNR expression and produce multi-fold greater levels of cellular dATP. Thus, we will test the dose dependence of elevated dATP for hiPSC-CMRNR engrafted into infarcted rat hearts. The novel aspect of our approach is to go beyond replacement of lost tissue (with hiPSC-CMs) by using engineered hiPSC-CMRNR to produce and deliver a small molecule therapeutic (dATP) that improves native heart muscle contraction. This has the potential to substantially recover the post-MI depressed function of native myocardium. Aim 2 will explore the mechanistic basis of how small increases in myocardial dATP result in significant increases in contractile force and kinetics of activation and relaxation of muscle, and in the magnitude of LV pressure development (LVDP) and kinetics of pressure development (+dP/dt) and decline (-dP/dt) of the heart. Our recent reports and preliminary data strongly suggest at least three mechanisms are involved: 1) disruption of the super-relaxed state (SRX) from the myosin backbone to a disordered relaxed state (DRX), 2) movement of DRX myosin towards thin filaments via greater electrostatic interactions with actin, and 3) faster crossbridge cycling. We have published multiple studies on the chemo-mechanics of faster crossbridge cycling (3), so will focus primarily on mechanisms 1 and 2 here using multiple state of the art approaches. These include low angle x-ray diffraction analysis of isolated myosin, cardiac muscle, and whole heart (Langendorff) levels, stopped-flow ATPase, super-localization single molecule microscopy of thick filament zones, structure-based computational models of myosin ± actin and multi-scale models of the heart. This project will elucidate the potential of our combination cell-small molecule therapy approach to improve function in failing hearts and provide understanding of the detailed molecular mechanisms of the myosin activator dATP.

抽象的。该项目的目标1围绕DRS之间的多年合作工作建立。雷格尼尔和穆里研究人类干细胞衍生的心肌细胞作为心脏修复的潜在细胞替代策略遵循心肌梗塞（MI）。我们已经表明，人类干细胞可以分化为心肌细胞（CMS），以规模和纯度产生，允许在啮齿动物模型和非人类中进行测试 Primes（NHP； Macaca Nemestrina），这些细胞植入并与宿主组织集成以改进左侧心室性能。拟议实验的前提是基于两个基本发现： 1）2-脱氧ATP（DATP）是心脏肌球蛋白使用时的潜在天然核核苷酸刺激剂，并且 2）hipsc-CM过表达DatP合成，核糖核苷酸还原酶（RNR），rnr）的速率限制酶增加了收缩性，还通过间隙连接将DATP运送到本地心肌心脏。在我们的当前奖项我们在测试以升高设计的HIPSC-CMS的假设方面取得了出色的进步 RNR（HIPSC-CMRNR）改善了MI细胞替代疗法的预后（与对照HIPSC-CMS相比）。对于此提案，我们已使用基因编辑的RNR生成了新的HIPSC-CM线条和不同的转录。发起人。这些细胞具有更大的RNR表达，并产生多重水平的细胞DATP。这是，我们将测试植入梗塞大鼠心脏的HIPSC-CMRNR升高的DatP的剂量依赖性。我们方法的新颖方面是通过使用而超越丢失的组织（使用HIPSC-CMS）。设计的HIPSC-CMRNR生产和提供小分子疗法（DATP），以改善本地心脏肌肉收缩。这有可能实质上恢复天然的MI后抑郁功能心肌。 AIM 2将探讨心肌datp少量增加的机械基础。收缩力和肌肉激活和放松的动力学显着增加 LV压力发展（LVDP）和压力发展的动力学（+DP/DT）和下降（-DP/DT）心。我们最近的报告和初步数据强烈建议至少涉及三种机制：1）从肌球蛋白骨架到无序的放松状态（DRX），2）的超浮雕状态（SRX）的破坏，2） DRX肌球蛋白通过与肌动蛋白的更大静电相互作用将DRX肌球蛋白移向细细丝，3）更快 Crossbridge骑自行车。我们已经发表了有关更快的Crossbridge骑行的化学力学的多项研究（3），因此主要将重点放在这里的机制1和2上，使用多种艺术方法。这些包括孤立肌球蛋白，心肌和全心（Langendorff）水平的低角度X射线衍射分析，停止流动ATPase，厚细丝区的超定位单分子显微镜，基于结构的基于结构肌球蛋白±肌动蛋白和心脏多尺度模型的计算模型。这个项目将阐明我们组合细胞小分子疗法的潜力，以改善失败心脏的功能和提供对肌球蛋白激活剂DatP的详细分子机制的理解。