Understanding the Mechanisms of Fibrosis in Muscular Dystrophies

项目总结 肌营养不良症(MD)是一种可遗传的神经肌肉疾病，可导致进行性虚弱和丧失。 肌肉的再生过程不能对进行性肌肉损伤作出充分反应。这最终是 结果用病理性细胞外基质(ECM)取代功能性肌肉组织 纤维化和脂肪，被称为纤维基质，这是最常见的MD的一个显著特征，Duchenne MD(DMD)。对抗纤维基质形成和促进肌肉再生的有效疗法是 MD患者的主要临床需求尚未得到满足，然而，导致这些问题的机制并不很好 明白了。该项目研究导致再生失败和纤维化的细胞机制。 营养不良肌肉的发育。具体地说，NAD(P)H氧化酶4(NOX4)已被鉴定为一种抗 肌肉中的纤维化和促再生靶点。肌肉纤维化的显著减少证明了这一点。 以及以NOX4为靶点的模拟DMD的严重营养不良肌肉的有益肌肉重构 使用遗传和药理学方法。推测NOX4在肌成纤维细胞、细胞中的表达 在组织损伤后产生细胞外基质，通过阻止肌成纤维细胞清除而促进肌肉纤维化 在肌肉再生之后。这种现象被称为肌成纤维细胞持续性。的实验。 本项目将严格研究导致这种疾病的机制和产生的病理后果。 通过使用创新的遗传模型、体外分析和 转录分析。目的1研究营养不良骨骼肌肌成纤维细胞的动力学 肌成纤维细胞命运图和NOX4的条件消融。肌成纤维细胞的持久性将在一项 新开发的使用营养不良肌肉分离细胞的检测方法，以及几种体外检测方法将被使用。 研究肌成纤维细胞在持续性分化和发展过程中的行为。AIM 2将评估 肌成纤维细胞对肌源性、成纤维细胞和成肌细胞基因表达和细胞行为的影响 营养不良和再生肌肉中的免疫细胞。体外模型将被用来区分物理和 这些肌成纤维细胞驱动表型的可扩散线索。当前项目的最终目标是 明确肌成纤维细胞在慢性肌肉疾病中的病理后果并提供可靠的 NOX4靶向作为有益重塑的有效影响的机制洞察力 治疗MDS的治疗策略，以及潜在的其他形式的肌肉病理。

PROJECT SUMMARY Muscular dystrophies (MDs) are heritable neuromuscular diseases that cause progressive weakness and loss of muscle as regenerative processes fail to adequately respond to progressive muscle damage. This ultimately results in the replacement of functional musculature with a pathological extracellular matrix (ECM) composed of fibrosis and fat, known as a fibro-matrix, which is a prominent feature of the most common of the MDs, Duchenne MD (DMD). Effective therapeutics to combat fibro-matrix development and facilitate muscle regeneration are a major unmet clinical need for MD patients, however, the mechanisms responsible for these issues are not well understood. This project investigates cellular mechanisms contributing to the failed regeneration and fibrosis development in dystrophic muscle. Specifically, NAD(P)H oxidase 4 (NOX4) has been identified as an anti- fibrotic and pro-regenerative target in muscle. This was demonstrated by marked reductions in muscle fibrosis and beneficial muscle remodeling of severely dystrophic muscle that models DMD, where NOX4 was targeted using genetic and pharmacological approaches. It is hypothesized that NOX4 expression in myofibroblasts, cells that produce ECM following tissue injury, contributes to muscle fibrosis by preventing myofibroblast clearance following muscle regeneration. This phenomenon is known as myofibroblast persistence. The experiments of this project will rigorously investigate the mechanisms leading to and the pathological consequences resulting from the development of myofibroblast persistence using innovative genetic models, in vitro assays, and transcriptomic analyses. Aim 1 will investigate myofibroblast dynamics in dystrophic skeletal muscle using myofibroblast fate-mapping and conditional ablation of NOX4. Myofibroblast persistence will be assessed in a newly-developed assay using cells isolated from dystrophic muscle, and several in vitro assays will be employed to investigate myofibroblast behavior during differentiation and development of persistence. Aim 2 will evaluate the influence that myofibroblasts exert on gene expression and cellular behavior of myogenic, fibroblastic, and immune cells in dystrophic and regenerating muscle. In vitro models will be used to discern physical versus diffusible cues responsible for these myofibroblast-driven phenotypes. The ultimate goal of the current project is to define the pathological consequences of myofibroblasts in chronic muscle disease and provide solid mechanistic insight responsible for the efficacious impact of NOX4-targeting as a beneficial remodeling therapeutic strategy for the treatment of MDs and, potentially, other forms of muscle pathology.