Regulating fibrosis and muscle growth in the muscular dystrophies

描述(申请人提供)：肌营养不良症是一种无法治愈的遗传性疾病。Duchenne肌营养不良症(DMD)是最严重的肌营养不良症之一。DMD和肢带型肌营养不良症的一部分共同破坏了肌营养不良蛋白复合体。破坏肌营养不良蛋白复合体会导致脆弱的肌膜，失去肌纤维，并以纤维化或疤痕取代肌肉。多条线的证据表明，纤维化是肌肉营养不良病理的驱动因素。我们假设，纤维化提供了一种支架，促进了不利的细胞因子谱，进一步损害了肌肉。我们进一步假设，不利的细胞因子谱的主要成分是TGFp及其相关的TGFp家族成员myostatin。TGFp和myostatin共同导致纤维化增加，肌肉质量和再生减少，并加重膜的脆性。因此，我们建议确定TGFp和myostatin通常被基质隔离而不能用于受体参与和信号传递的方式，并确定如何在肌营养不良症中促进TGFp和myostatin的失活(项目1)。我们还将演示释放和处理肌肉生长抑素及其相关分子所需的蛋白分解步骤，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TGFp和myostatin触发的不同的细胞内信号通路，并测试抑制这些通路是否改善了肌营养不良症的肌肉功能和病理(项目3)。三位资深研究人员(McNally、Lee和Molkentin)将领导这些项目，组成一个独特的团队，他们的专业知识将共同定义肌营养不良症治疗意图的TGFp/myostatin途径。三个核心将支持这些项目；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让。核心B将对基因操作和治疗后的肌营养不良症进行组织病理学评估，核心C将在体内进行功能分析，并为核心B提供支持。 与公共卫生相关：肌肉营养不良是一种破坏性的疾病，影响儿童和成人，目前还没有治愈的方法。我们现在知道，疤痕在肌肉营养不良症的肌肉无力中扮演着重要的角色。肌肉营养不良研究领域的三位主要研究人员将协同设计和实施治疗这种疾病的新战略。我们的重点是TGPp和myostatin调节的通路，以及操纵这一通路以减少疤痕、促进生长和促进肌肉稳定的目标。 项目1 主要调查者：伊丽莎白·M·麦克纳利，医学博士 标题：LTBPS作为转化生长因子和肌肉生长抑素的调节因子 描述(由申请人提供)：营养不良蛋白复合体的破坏会导致肌肉膜变得脆弱，极易受到损害。肌营养不良症的定义是肌肉持续退化并伴有再生不足。虽然肌肉再生在这些疾病中正在进行，但它是无效的，纤维脂肪渗透最终取代了肌肉，导致肌肉损失和虚弱。我们最近使用了一种肢体Girdle肌营养不良症(LGMD)的小鼠模型，即缺乏y-肌聚糖的SGcg缺失小鼠，以探讨遗传修饰基因是否可以改变肌营养不良症的结果。我们使用肌肉病理的两个定量评估来衡量结果，埃文斯蓝染料摄取(染料摄取)用于测量膜的脆性和渗漏，胶原沉积用于反映肌肉中的瘢痕形成。我们发现，膜的脆性和瘢痕形成都被编码潜在的TGFb结合蛋白的基因Ltbp4强烈地修饰。遗传和分子数据支持LTBP4的裂解释放TGFb，使其更容易被肌肉内的细胞所利用。增强的TGFp信号促进了肌营养不良症的膜破裂和瘢痕形成。类似地，全长LTBP4的增加可以保护膜渗漏和纤维化。基于对相关LTBP家族成员的研究，我们假设LTBP4也与肌肉生长抑素结合。我们假设了一个肌营养不良发病机制的模型，其中LTBP4裂解释放了myostatin和TGFb，从而通过增加疤痕和减少肌肉生长来促进肌肉营养不良。在目标1中，我们将通过在肌纤维中过度表达LTBP4的保护形式来检验这一假设，并确定这是否减少了肌肉营养不良和下游信号转导。在目标2中，我们将确定LTBP4是否与myostatin结合，以及LTBP4通过myostatin通过myostatin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LTBP4，因为很可能肌肉抑制素和LTBP4都被相同的蛋白酶激活。信号方面和蛋白水解酶实验将与项目2和项目3一起进行。 与公共健康相关：麦克纳利实验室最近发现了一种名为Ltbp4的基因，它可以改变肌营养不良症的结果。我们现在将使用增加LTBP4的技术来测试这种基因发现，并确定它是否通过Myostatin和TGFp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.