Regulating fibrosis and muscle growth in the muscular dystrophies

描述（由申请人提供）：肌营养不良是一种遗传性疾病，目前尚无治愈方法。肌营养不良症的最严重形式之一是杜氏肌营养不良症（DMD）。DMD和肢带型肌营养不良症的一个亚群共同具有肌营养不良蛋白复合物的破坏。破坏抗肌萎缩蛋白复合物导致脆弱的肌肉膜、肌纤维损失和肌肉纤维化或瘢痕形成。多种证据表明纤维化是肌营养不良病理学的驱动因素。我们假设纤维化提供了一个支架，促进不利的细胞因子谱，进一步损害肌肉。我们进一步假设，不利的细胞因子谱的主要成分是TGF β和相关的TGF β家族成员肌肉生长抑制素。TGF β和肌生长抑制素一起导致纤维化增加、肌肉质量和再生减少以及膜脆性加剧。因此，我们建议确定TGF β和肌肉生长抑制素通常被基质隔离并保持不可用于受体接合和信号传导的方式，并确定如何促进肌营养不良症中TGF β和肌肉生长抑制素的失活（项目1）。我们还将证明肌肉生长抑制素和相关分子的释放和加工所需的蛋白水解切割步骤，以及可溶性受体在治疗肌营养不良症中的有效程度（项目2）。我们还将依次评估由TGF β和肌生长抑制素触发的不同细胞内信号传导途径，并测试抑制这些途径是否改善肌营养不良症的肌肉功能和病理学（项目3）。三位已建立的研究人员（McNally，Lee和Molkentin）将领导这些项目，形成一个独特的团队，他们的综合专业知识将定义肌营养不良症治疗意图的TGF β/肌肉生长抑制素途径。三个核心将支持项目;核心A将整合我们三个机构的努力，以确保无缝协作和材料转移。核心B将在基因操作和治疗后提供肌营养不良症的组织病理学评估，核心C将进行体内功能分析并为核心B提供支持。 公共卫生相关性：肌营养不良是一种影响儿童和成人的毁灭性疾病，目前尚无治愈方法。我们现在了解到，瘢痕形成在肌营养不良症的进一步肌无力中起着重要作用。三个首屈一指的研究人员在肌肉萎缩症的研究领域将协同设计和实施新的战略来治疗这种疾病。我们的重点是TGPp和肌肉生长抑制素调控的途径，以及操纵该途径以减少瘢痕形成，增加生长和促进肌肉稳定性的目标。 项目1 主要研究者：Elizabeth M.麦克纳利，医学博士，博士 标题：LTBPS作为TGF β和肌生长抑制素的调节剂 描述（由申请方提供）：抗肌萎缩蛋白复合物的破坏导致肌肉膜变得脆弱且极易受损。肌营养不良的定义是持续的肌肉退化与再生不足相结合。虽然肌肉再生在这些疾病中正在进行，但它是无效的，纤维脂肪浸润最终取代肌肉，导致肌肉损失和虚弱。我们最近使用了肢带型肌营养不良症（LGMD）的小鼠模型，即缺乏γ-肌聚糖的Sgcg基因缺失小鼠，以询问遗传修饰基因是否可以改变肌营养不良症的结果。我们使用肌肉病理学的两种定量评估来测量结果，伊文思蓝染料摄取（染料摄取）用于测量膜脆性和渗漏，胶原蛋白沉积用于反映肌肉中的瘢痕形成。我们发现，这两个膜脆性和疤痕都强烈修改Ltbp 4，基因编码的潜伏性TGFB结合蛋白。遗传和分子数据支持LTBP 4的切割释放TGFB，使其更容易被肌肉内的细胞利用。增强的TGF β信号传导促进肌营养不良症中的膜破坏和瘢痕形成。类似地，全长LTBP 4的增加对膜渗漏和纤维化都有保护作用。基于对相关LTBP家族成员的研究，我们假设LTBP 4也结合肌生长抑制素。我们提出了一个肌营养不良发病机制模型，其中LTBP 4切割释放肌生长抑制素和TGFB，从而通过增加疤痕和减少肌肉生长来促进肌营养不良。在目标1中，我们将通过在肌纤维中过表达LTBP 4的保护形式来测试这一假设，并确定这是否会减少肌营养不良和下游信号传导。在目的2中，我们将使用肌生长抑制素缺失小鼠确定LTBP 4是否结合肌生长抑制素以及LTBP 4通过肌生长抑制素介导其作用的程度。在目标3中，我们将评估哪些蛋白酶切割LTBP 4，因为肌生长抑制素和LTBP 4两者很可能被相同的蛋白酶激活。信号方面和蛋白酶实验将与项目2和项目3一起进行。 公共卫生相关性：McNally实验室最近发现了一种名为Ltbp 4的基因，可以改变肌肉萎缩症的结果。我们现在将使用增加LTBP 4的技术来测试这一遗传发现，并确定它是否通过肌肉生长抑制素和TGF β发挥作用。

DESCRIPTION (provided by applicant): Muscular dystrophy is a genetic disease for which there is no cure. One of the most severe forms of muscular dystrophy is Duchenne Muscular Dystrophy (DMD). DMD and a subset of the limb girdle muscular dystrophies have in common disruption of the dystrophin protein complex. Disrupting the dystrophin complex lead to a fragile muscle membrane, loss of myofibers and replacement of the muscle with fibrosis or scarring. Multiple lines of evidence point to fibrosis is as a driver of muscular dystrophy pathology. We hypothesize that fibrosis provides a scaffold that promotes an unfavorable cytokine profile that further damages muscle. We further hypothesize that the primary components of the unfavorable cytokine profile are TGFp and the related TGFp family member myostatin. Together, TGFp and myostatin, lead to increased fibrosis, reduced muscle mass and regeneration, and aggravated membrane fragility. Therefore, we propose to determine the means by which TGFp and myostatin are normally sequestered by the matrix and held unavailable for receptor engagement and signaling and to determine how to promote inactivation of TGFp and myostatin in muscular dystrophy (Project 1). We will also demonstrate necessary proteolytic cleavage steps for release and processing of myostatin, and related molecules, and the degree to which soluble receptors can be effective in treating muscular dystrophy (Project 2). We will also sequentially assess the distinct intracellular signaling pathways that are triggered by TGFp and myostatin and test whether inhibiting these pathways improves muscle function and pathology in muscular dystrophy (Project 3). Three established investigators (McNally, Lee, and Molkentin) will lead these projects forming a distinctive team where their combined expertise will define the TGFp/myostatin pathway for therapeutic intent in muscular dystrophy. Three Cores will support the Projects; Core A will integrate the efforts at our three institutions to assure seamless collaboration and transfer of materials. Core B will provide histopathological assessment of muscular dystrophy after genetic manipulation and treatments, and Core C will perform functional analysis in vivo and provide support to Core B. PUBLIC HEALTH RELEVANCE: Muscular dystrophy is a devastating disorder that affects children and adults and for which there is no cure. We now understand that scarring plays an important role in furthering muscle weakness in the muscular dystrophies. Three premier investigators in the field of muscular dystrophy research will synergistically devise and implement new strategies to treat this disease. Our focus is on a pathway regulated by TGPp and myostatin and the goals of manipulating this pathway to reduce scarring, increase growth and promote muscle stability. PROJECT 1 Principal Investigator: Elizabeth M. McNally, M.D., Ph.D. Title: LTBPS as Regulators of TGF¿ and Myostatin Description (provided by applicant): Disruption of the dystrophin complex causes the muscle membrane to become fragile and highly susceptible to damage. Muscular dystrophy is defined by ongoing muscle degeneration combined with insufficient regeneration. Although muscle regeneration is ongoing in these disorders, it is ineffective and fibrofatty infiltration ultimately replaces muscle resulting in muscle loss and weakness. We recently used a mouse model of Limb Girdle Muscular Dystrophy (LGMD), the Sgcg null mouse lacking y-sarcoglycan, to ask whether genetic modifier genes can alter the outcome in muscular dystrophy. We measured outcome using two quantitative assessments of muscle pathology, Evans blue dye uptake (dye uptake) to measure membrane fragility and leakiness and collagen deposition to reflect scarring in the muscle. We found that both membrane fragility and scarring were both strongly modified by Ltbp4, the gene encoding the latent TGFB binding protein. Genetic and molecular data support that cleavage of LTBP4 releases TGFB, making it more available to the cells within muscle. Enhanced TGFp signaling promotes both membrane disruption and scarring in muscular dystrophy. Similarly, an increase in full length LTBP4 is protective of both membrane leakiness and fibrosis. Based on studies performed with the related LTBP family members, we hypothesize that LTBP4 also binds myostatin. We posit a model for muscular dystrophy pathogenesis where LTBP4 cleavage releases both myostatin and TGFB, thereby promoting muscular dystrophy through increased scarring and decreased muscle growth. In Aim 1, we will test this hypothesis by overexpressing the protective form of LTBP4 in myofibers and determining whether this reduces muscular dystrophy and downstream signaling. In Aim 2, we will determine whether LTBP4 binds myostatin and the degree to which LTBP4 mediates its effects through myostatin using myostatin null mice. In Aim 3, we will assess which proteases cleave LTBP4 since it is likely that both myostatin and LTBP4 are activated by the same proteases. The signaling aspects and protease experiments will be conducted in conjunction with Projects 2 and Project 3. Public Health Relevance: The McNally laboratory recently discovered a gene, called Ltbp4, that can modify the outcome of muscular dystrophy. We will now test that genetic discovery using techniques to increase LTBP4 and to determine whether it exerts its effects through myostatin as well as TGFp.