Engineered Stem Cells for Cardiac Repair

抽象的。该项目的目标1建立在Regnier博士和Murry博士多年的合作基础上 研究人干细胞来源的心肌细胞作为心脏修复的潜在细胞替代策略 心肌梗死(MI)后。我们已经证明，人类干细胞可以分化为 心肌细胞(CMS)，其培养规模和纯度允许在啮齿动物模型和非人类模型中进行测试 灵长类动物(NHP；猕猴)，这些细胞植入并与宿主组织整合以改善左侧 脑室功能。拟议实验的前提是基于两个基本发现： 1)2-脱氧三磷酸腺苷(DATP)是心肌肌球蛋白使用时一种有效的天然核苷酸收缩刺激剂，以及 2)过表达dATP合成限速酶、核糖核苷酸还原酶(RNR)、 增加收缩能力，并通过缝隙连接将dATP输送到自然心肌。在我们的 目前的奖项，我们在检验假设方面取得了很好的进展，该假说是用提升的 RnR(hiPSC-CMRNR)可改善心肌梗死细胞替代治疗的结果(与对照hiPSC-CMS相比)。 对于这一建议，我们已经创建了具有基因编辑的RNR和不同转录水平的新的HiPSC-CM系 推动者。这些细胞具有更高的RNR表达，并产生多倍于细胞内dATP的水平。 因此，我们将测试hPSC-CMRNR植入梗死大鼠心脏后dATP升高的剂量依赖性。 我们方法的新方面是超越通过使用hPSC-CMS来替换丢失的组织 工程HiPSC-CMRNR生产和提供一种改善天然心脏的小分子治疗(DATP) 肌肉收缩。这有可能实质上恢复心肌梗塞后抑郁的功能。 心肌。目标2将探索心肌dATP小幅增加如何导致 显著增加肌肉的收缩力量和激活和放松的动力学，并在幅度上 左心室压力发展(LVdP)和压力发展(dp/dt)和下降(-dp/dt)的动力学 心。我们最近的报告和初步数据强烈表明，至少有三种机制参与其中：1) 从肌球蛋白骨架的超松弛状态(SRX)到无序松弛状态(DRX)的破坏，2) DRX肌球蛋白通过与肌动蛋白更大的静电相互作用向细丝移动，以及3)更快 跨桥自行车赛。我们已经发表了多项关于更快的过桥自行车的化学机制的研究 (3)，在这里，SO将使用多种最先进的方法主要集中在机制1和2上。这些措施包括 分离肌球蛋白、心肌和全心(朗宁多夫)水平的低角x射线衍射分析， 停流ATPase，粗丝区的超定位单分子显微镜，基于结构 肌球蛋白±肌动蛋白的计算模型和心脏的多尺度模型。这个项目将阐明 我们的细胞-小分子联合疗法在改善衰竭心脏和心脏功能方面的潜力 了解肌球蛋白激活剂dATP的详细分子机制。

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.