Engineering a Microphysiological System to Model the Infarct Border Zone and Interrogate Oxygen-Dependent Cell-Cell Communication in the Myocardium

项目摘要/摘要 心肌梗死(MI)是心脏损伤最常见的形式之一。在心肌梗塞中，冠状动脉 闭塞导致局部限制心肌的血液和氧气供应，导致许多即刻 以及整个心肌的长期变化，通常表现为心律失常或心力衰竭。因此，一个 对发生在局部和远端缺氧损伤的重塑过程的更详细的理解是 迫切需要开发新的治疗方法来减缓心肌梗死后心力衰竭的进展。近期 研究表明，缺氧改变了心脏成纤维细胞和心脏成纤维细胞分泌的外切体中的货物。 肌细胞。这表明，缺氧和正常缺氧的心肌细胞类型可以通过外切体在 心肌梗死后心肌。低氧心肌细胞分泌的外切体对常氧心脏的影响 细胞(反之亦然)的特性很差，这在很大程度上是由于缺乏实验工具。例如， 研究体外缺氧的最新技术是通过培养箱或低氧来调节全局氧气 密室。这种方法不能模拟心肌梗死后心肌的特征氧梯度 因此排除了对常氧和低氧之间正在进行的细胞间通讯的研究 细胞，这可能是心肌梗死后心肌重构的关键机制。我们假设本地化 缺氧对心肌细胞表型的影响，局部是由于氧的直接作用，远端是由于 低氧和常氧细胞之间的细胞间通讯，主要由外切体介导。为了测试这一点 假设，我们将首先制造一个新的微生理系统，它：(1)实现微流控供气 产生氧气梯度的通道；(2)具有模块化的细胞培养室，以调节细胞与细胞的接触和 旁分泌信号；以及(3)整合定量心脏成纤维细胞和心肌细胞结构和 功能表型，包括现有的“芯片上的心脏”收缩能力分析，以前由PI提出。 然后，我们将为三个目标实施这些设备。在目标1中，我们将测量氧气梯度如何影响 心脏成纤维细胞表型、外切体RNA和蛋白质含量以及氧敏感活性 转录因子，HIF-1和HIF-2。在目标2中，我们将对心肌细胞和 通过整合我们用于测量传播速度的“芯片上的心脏”分析来量化功能表型 和收缩压力。在目标3中，我们将描述低氧成纤维细胞和常氧成纤维细胞之间的串扰。 心肌细胞，以及低氧心肌细胞和常氧成纤维细胞。共同努力，我们的创新 微生理系统和严格的实验方法将揭示对 局部缺氧损伤对心肌细胞表型的影响及其与心肌损伤的关系 心肌梗塞后的重塑。我们的数据还将建立与细胞-细胞通讯通路相关的新范式 低氧是由外切体介导的，它可以在治疗上发挥杠杆作用。此外，我们的新产品 该设备可用于对减轻低氧影响的化合物进行中通量筛选。

PROJECT SUMMARY/ABSTRACT Myocardial infarction (MI) is one of the most common forms of cardiac injury. In a MI, coronary artery occlusion leads to a local restriction of blood and oxygen supply to the myocardium, causing many immediate and long-term changes throughout the myocardium that often manifest as arrhythmias or heart failure. Thus, a more detailed understanding of the remodeling processes that occur both local and distant to hypoxic injury are critically needed to develop new therapies for mitigating the progression to heart failure after MI. Recent studies have shown that hypoxia alters the cargo found in exosomes secreted by both cardiac fibroblasts and myocytes. This suggests that hypoxic and normoxic cardiac cell types could communicate via exosomes in post-MI myocardium. However, the effects of exosomes secreted by hypoxic cardiac cells on normoxic cardiac cells (and vice versa) is poorly characterized, in large part due to a lack of experimental tools. For example, the state-of-the-art for investigating hypoxia in vitro is to modulate oxygen globally with an incubator or hypoxia chamber. This approach does not mimic the oxygen gradients that are characteristic of post-MI myocardium and therefore precludes the investigation of ongoing cell-cell communication between normoxic and hypoxic cells, which could be a key mechanism of myocardial remodeling post-MI. We hypothesize that localized hypoxia affects the phenotypes of cardiac cells locally due to the direct effects of oxygen, and distally due to cell-cell communication between hypoxic and normoxic cells, mediated primarily by exosomes. To test this hypothesis, we will first fabricate a new microphysiological system that: (1) implements microfluidic gas supply channels to generate oxygen gradients; (2) has modular cell culture chambers to regulate cell-cell contact and paracrine signaling; and (3) integrates assays for quantifying cardiac fibroblast and myocyte structural and functional phenotypes, including existing “Heart on a Chip” contractility assays previously advanced by the PI. We will then implement these devices for three Aims. In Aim 1, we will measure how oxygen gradients affect cardiac fibroblast phenotype, exosome RNA and protein cargo, and the activity of the oxygen-sensitive transcription factors, HIF-1 and HIF-2. In Aim 2, we will perform similar studies with cardiac myocytes and quantify functional phenotypes by integrating our “Heart on a Chip” assays for measuring propagation velocity and contractile stress. In Aim 3, we will characterize cross-talk between hypoxic fibroblasts and normoxic cardiac myocytes, as well as hypoxic cardiac myocytes and normoxic fibroblasts. Together, our innovative microphysiological systems and rigorous experimental approaches will reveal significant new insights into the effects of localized hypoxic injury on the phenotypes of cardiac cell types, relevant to understanding myocardial remodeling post-MI. Our data will also establish new paradigms related to cell-cell communication pathways in hypoxia that are mediated by exosomes, which could be leveraged therapeutically. Additionally, our new devices could be used for medium-throughput screening of compounds for mitigating the effects of hypoxia.