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

项目总结/摘要 心肌梗死（MI）是最常见的心脏损伤形式之一。心肌梗塞时，冠状动脉 闭塞导致心肌的血液和氧气供应的局部限制， 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 更详细地了解缺氧损伤局部和远处发生的重塑过程， 迫切需要开发新的治疗方法来缓解MI后心力衰竭的进展。最近 研究表明，缺氧改变了由心脏成纤维细胞分泌的外泌体中发现的货物， 肌细胞这表明缺氧和常氧心肌细胞类型可以通过外泌体进行通讯， MI后心肌然而，低氧心肌细胞分泌的外泌体对常氧心肌细胞的影响是不稳定的。 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。比如说 研究体外缺氧的现有技术是用培养箱或缺氧器全面调节氧气 室。这种方法不能模拟心肌梗死后心肌的氧梯度特征 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塑的关键机制。我们假设， 缺氧局部影响心肌细胞的表型，这是由于氧的直接作用，而远端影响心肌细胞的表型， 低氧和常氧细胞之间的细胞间通讯，主要由外来体介导。为了验证这一 假设，我们将首先制造一个新的微生理系统，其：（1）实现微流体气体供应 （2）具有模块化细胞培养室以调节细胞-细胞接触， 旁分泌信号传导;和（3）整合用于定量心脏成纤维细胞和肌细胞结构和 功能表型，包括PI先前提出的现有“芯片上的心脏”收缩性测定。 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，外泌体RNA和蛋白质货物，以及氧敏感的活性 转录因子HIF-1和HIF-2。在目标2中，我们将对心肌细胞进行类似的研究， 通过整合我们用于测量传播速度的“芯片上的心脏”测定来量化功能表型 和收缩应力。在目标3中，我们将描述缺氧成纤维细胞和常氧成纤维细胞之间的串扰。 心肌细胞以及缺氧心肌细胞和常氧成纤维细胞。我们的创新 微生理系统和严格的实验方法将揭示重要的新见解， 局部缺氧损伤对心肌细胞表型的影响，与了解心肌细胞相关 心肌梗死后重塑我们的数据还将建立与细胞间通讯途径相关的新范式， 缺氧是由外来体介导的，这可以在治疗上加以利用。此外，我们的新 该装置可用于中等通量筛选减轻缺氧影响的化合物。

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.