Can klotho alleviate muscle fibrosis in muscular dystrophy?

描述(申请人提供)：Duchenne肌营养不良症(DMD)与营养不良蛋白基因突变有关，导致横纹肌进行性萎缩和过早死亡。DMD患者的骨骼肌经历肌纤维坏死和再生的循环，但随着时间的推移，肌纤维丢失，退化的肌纤维被纤维组织取代。治疗DMD需要稳定肌纤维肌膜和抗击纤维化的方法。虽然已经对这两个方面进行了深入的研究，但有效的治疗方法仍有待开发。这项拟议的研究将分析Klotho(也称为α-Klotho)在dystrophin缺失的MDX小鼠中减少纤维化的潜力，这是一种肌肉营养不良的小鼠模型。Klotho既是一种跨膜蛋白，又是一种分泌型体液因子。分泌的Klotho已被证明直接抑制肾组织中促纤维化的转化生长因子-β1和Wnt途径。虽然Klotho的主要表达部位是肾脏，但它也在其他组织中表达。如果Klotho的抗纤维化作用扩展到肌肉组织，Klotho蛋白可能成为一个有趣的候选方案，用于减少肌营养不良症患者的肌肉纤维化，目前已经确定了转化生长因子-β1和Wnt在肌肉纤维化中的作用。拟议项目的一个主要目的是深入了解Klotho转基因(Kt)过表达缓解mdx小鼠肌肉纤维化的潜力。除了纤维化、钙化和肌纤维病理的组织学分析外，这项研究还将检测CD45+细胞水平，以此作为衡量炎症和似乎受到纤维化损害的肌源性(卫星细胞)活动的指标。第二个目的是通过检测在MDX小鼠中自愿车轮运动的下降来分析Kt转基因对全身表现的影响。将使用mdx4cv品系，它比标准的mdx小鼠品系产生的回复肌纤维更少。重点将给予MDX和Kt/MDX小鼠从年轻到老年的分组，以获得随着MDX肌肉病理随年龄增长而加剧的全面洞察。拟议研究的小鼠品系和专业知识都已可用，从而能够取得有效进展。人们认识到，虽然MDX小鼠已被广泛用作DMD的实验室模型，但MDX的病理不如DMD患者严重。尽管如此，对MDX小鼠的研究已经为随后的营养不良狗研究和人类临床试验提供了重要的指导数据。在拟议的研究中额外包括野生型(Wt)和Kt/wt小鼠群体，不仅可以与亲本进行比较，还可以深入了解Klotho转基因在野生型背景下的影响。这项探索性研究的结果可能为对抗DMD和其他肌肉营养不良以及改善衰老过程中的肌肉质量确定一个新的临床方向。旨在鼓励探索性、高风险、高回报的新奇研究的R21机制是这一应用的最佳平台。

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is associated with mutations in the dystrophin gene, leading to progressive wasting of striated muscle and early death. Skeletal muscles in DMD patients undergo cycles of myofiber necrosis and regeneration, but over time there is a loss of myofibers and the degenerating myofibers are replaced with fibrotic tissue. Treating DMD requires means for stabilizing the myofiber sarcolemma and for combating fibrosis. Although both fronts have been intensely studied, effective treatments are yet to be developed. The proposed study will analyze the potential of klotho (also known as alpha-klotho) to reduce fibrosis in the dystrophin-null mdx mouse, a murine model of muscular dystrophy. Klotho functions as both a transmembrane protein and a secreted humoral factor. The secreted klotho has been shown to directly inhibit the pro-fibrotic TGF-ß1 and Wnt pathways in renal tissue. While the main site of klotho expression is the kidney, it is also expressed in other tissues. Should the anti-fibrotic effect of klotho extend to he muscle tissues, the klotho protein could become an intriguing candidate for reducing muscle fibrosis in dystrophin-deficiency where a role for TGF-ß1 and Wnt in muscle fibrosis has been established. One main aim of the proposed project is to gain insight into the potential of klotho transgenic (Kt) overexpression to alleviate muscle fibrosis in mdx mice. In addition to histological assays of fibrosis, calcification and myofiber pathology, the study will examine CD45+ cell level as a measure of inflammation and myogenic (satellite cell) activity that appears to be impaired by fibrosis. A second aim is to analyze the effect of the Kt transgene on whole body performance by examining voluntary wheel running, which declines in the mdx mouse. The mdx4cv strain that develops less revertant myofibers than the "standard" mdx mouse line will be used. Emphasis will be given to mdx and Kt/mdx mouse groups of young to old ages to gain a comprehensive insight as the mdx muscle pathology intensifies with age. Both the mouse lines and the expertise for the proposed studies are already available, permitting effective progress. It is recognized that while the mdx mouse has been used extensively as a laboratory model of DMD, the mdx pathology is less severe than that of DMD patients. Still, studies with mdx mice have generated important directive data for subsequent studies with dystrophic dogs and for pilot clinical trials in humans. The additional inclusion of wildtype (wt) and Kt/wt mouse groups in the proposed study will not only permit comparison with the parental lines, but also allow gaining insights into the effect of the klotho transgene in the wildtype context. The outcome of this exploratory study may identify a new clinical direction for combating DMD and other muscular dystrophies as well as improving muscle quality in aging. The R21 mechanism that is intended to encourage exploratory high-risk high-reward novel studies is the optimal platform for this application.