Developing An In Vitro Human Myobundle Model Of Rheumatoid Arthritis

摘要 风湿性关节炎（RA）是一种慢性炎症性疾病，主要影响关节。自身反应 免疫细胞进入关节和骨骼肌，滑膜炎症 损伤软骨引起疼痛和不适。5至10年的RA残疾率达到40% 诊断后。除了疾病活动和疼痛，肌肉力量对残疾有重大影响， RA患者。骨骼肌显然有助于类风湿关节炎的残疾，但发生这种情况的机制是 没有被很好地理解。由于动物模型和细胞培养系统是人类疾病的不完美复制品， 不能概括人体骨骼肌的生物学和生理学特征，我们建议使用 最近开发的工程化电响应，收缩人类肌肉组织（肌束）， 在体外建立RA模型并鉴定疾病表型的特征。这些人类肌束呈现出 天然骨骼肌的标志，包括对齐的结构、多核和横纹肌纤维，以及 卫星细胞池培养四周后，它们自发收缩，并对单一或高浓度的 频率电刺激伴抽搐和强直收缩;肌束维持功能性乙酰胆碱 和β-肾上腺素能受体，并经历结构和功能成熟。成肌细胞的初步数据 来自RA患者的研究表明，RA降低了肌纤维形成和产生显著 收缩力，甚至在去除多余的成纤维细胞后。建立体外系统作为检测模型 治疗骨骼肌疾病，我们将扩展我们的初步结果，并测试假设 假设（1）从类风湿性关节炎患者的细胞衍生的工程化骨骼肌肌束 表现出减少的修复和分化能力以及减少的力量生产;（2） 分化和功能是由于选择肌因子和细胞因子产生;和（3）添加细胞因子和 由RA骨骼肌肌纤维分泌的肌因子可在健康成肌细胞中诱导RA表型。在 目的1，我们将扩展我们的初步结果，表明工程化骨骼肌肌束， 复制疾病表型并表现出降低的成熟和力生成能力。 用来自健康年龄匹配个体的成肌细胞制备的肌束将作为对照。结果 将与活检样本的免疫组织化学染色进行比较，以确定RA中的细胞变化 肌肉.在目标2中，我们将使用无偏的方法并测量RA释放的蛋白质和细胞因子 并应用统计模型来确定RA表型特征之间的关系 目的1中鉴定的细胞因子和细胞因子的水平。在目标3中，我们将假设 RA肌肉的肌因子和细胞因子的异常分泌是疾病表型的原因。

Abstract Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects the joints. Autoreactive immune cells enter articular joints as well as skeletal muscle, and inflammation of the synovial membrane damages the cartilage causing pain and discomfort. Disability rates from RA reach 40% from 5 to 10 years after diagnosis. In addition to disease activity and pain, muscle strength has a major impact on disability in RA patients. Skeletal muscle clearly contributes to RA disability, but the mechanisms by which this occurs are not well understood. Since animal models and cell culture systems are imperfect replicates of human disease and do not recapitulate biologic and physiologic features of human skeletal muscle, we propose to use a recently developed engineered electrically responsive, contractile human muscle tissues (myobundles) to model RA in vitro and identify features of the disease phenotype. These human myobundles exhibit structural hallmarks of native skeletal muscle including aligned architecture, multinucleated and striated myofibers, and a satellite cell pool. Over four weeks in culture, they contract spontaneously and respond to single or high frequency electrical stimuli with twitch and tetanic contractions; myobundles maintain functional acetylcholine and β-adrenergic receptors and undergo structural and functional maturation. Preliminary data with myoblasts derived from RA patients indicate that RA reduces the ability of myofibers to form and produce significant contractile force, even after removal of excess fibroblasts. To establish the in vitro system as a model to test therapies for the disease in skeletal muscle, we will extend our preliminary results and test the hypotheses hypotheses that (1) engineered skeletal muscle myobundles derived from cells of rheumatoid arthritis patients show reduced capacity for repair and differentiation and reduced force production; (2) the decrement in differentiation and function is due to select myokine and cytokine production; and (3) addition cytokines and myokines secreted by RA skeletal muscle myofibers can induce the RA phenotype in healthy myoblasts. In Aim 1, we will extend our preliminary results to show that the engineered skeletal muscle myobundles reproduce the disease phenotype and exhibit reduced maturation and capacity for force generation. Myobundles prepared with myoblasts from healthy aged-matched individuals will serve as controls. Results will be compared to immunohistochemical staining of biopsy samples to establish cellular changes in RA muscle. In Aim 2, we will use an unbiased approach and measure proteins and cytokines released from RA myobundles and apply statistical model to determine the relationship between features of RA phenotype identified in Aim 1 and the levels of myokines and cytokines. In Aim 3, we will that the hypothesis that the abnormal secretion of myokines and cytokines by RA muscle is responsible for the disease phenotype.