Mechanistic basis and potential therapies for myosin storage myopathy

描述（由申请人提供）：我们建议建立并分析第一个报道的肌球蛋白储存性肌病（MSM）动物模型，这是一种人类骨骼和心肌的退行性疾病，由慢/β-心肌肌球蛋白重链 C 端杆区的点突变引起。我们的转基因黑腹果蝇模型将用于剖析 MSM 的分子和发育基础并测试可能的治疗方式。果蝇系统将使我们能够在标准化遗传背景下检查每个 MSM 等位基因的纯合子和杂合子（突变体/）。这将确定野生型和突变型肌球蛋白分子之间相互作用对疾病病理学的重要性，并将消除导致人类疾病表型变异的遗传异质性。我们将测试以下假设：1) 果蝇中表达的 MSM 突变肌球蛋白导致与人类 MSM 患者相似的特定细胞生物学和生理异常，2) MSM 肌球蛋白分子 a) 肌丝组装有缺陷，b) 显示异常肌球蛋白降解和/或 c) 易于聚集，3) 阻止 MSM 肌球蛋白聚集体形成或增强 MSM 肌球蛋白更新可以改善突变肌肉结构和性能。为了检验这些假设，我们将追求以下具体目标：1）检查衰老过程中四种不同的 MSM 突变对骨骼肌和心肌的结构和功能影响。我们将通过显微镜和生理测定探索 MSM 的渐进性质，并将我们的结果与现有的人类数据相关联。 2) 分离突变肌球蛋白分子并评估其丝形成能力、丝对蛋白水解的稳定性和聚集倾向。这将有助于确定该疾病的分子基础。 3) 尝试改善生物体中的疾病表型 a) 通过过度表达分子伴侣 αB-晶状体蛋白、Hsp70、Hsp90 或 UNC-45（均已知有助于肌球蛋白折叠和/或免受应激）； b) 通过使用热休克反应的小分子诱导剂来更广泛地引发分子伴侣的表达； c) 通过使用转基因或药理学方法诱导自噬作为清除肌球蛋白聚集体的机制。这种多方面的方法将为 MSM 的发育和生物物理基础提供新的见解，并产生可能有助于治疗 MSM 和其他包涵体疾病的潜在治疗方法。

DESCRIPTION (provided by applicant): We propose to build and analyze the first reported animal models of myosin storage myopathy (MSM), a degenerative disease of human skeletal and cardiac muscles that arises from point mutations in the C-terminal rod region of slow/beta-cardiac myosin heavy chain. Our transgenic Drosophila melanogaster models will be used to dissect the molecular and developmental bases of MSM and to test possible therapeutic modalities. The Drosophila system will allow us to examine both homozygotes and heterozygotes (mutant/+) for each MSM allele in a standardized genetic background. This will define the importance of interactions between wild- type and mutant myosin molecules to disease pathology and will obviate genetic heterogeneity that leads to phenotypic variability in the human disease. We will test the hypotheses that: 1) MSM mutant myosin expressed in Drosophila leads to specific cell biological and physiological abnormalities similar to those seen in human MSM patients, 2) MSM myosin molecules a) are defective in filament assembly, b) show abnormal filament degradation and/or c) are prone to aggregation, 3) preventing MSM myosin aggregate formation or enhancing MSM myosin turnover can improve mutant muscle structure and performance. To test these hypotheses, we will pursue the following specific aims: 1) Examine the structural and functional effects of four different MSM mutations on skeletal and cardiac muscles during aging. We will explore the progressive nature of MSM via microscopy and physiological assays and correlate our results with extant human data. 2) Isolate mutant myosin molecules and assess their filament-forming ability, filament stability to proteolysis and aggregation propensity. This will help define the molecular basis of the disease. 3) Attempt to ameliorate disease phenotypes in organisms a) by over-expressing the molecular chaperones alphaB-crystallin, Hsp70, Hsp90 or UNC-45 (all known to aid in myosin folding and/or protection from stress); b) by using small molecule inducers of the heat shock response to more broadly elicit expression of molecular chaperones; c) by using transgenic or pharmacological approaches to induce autophagy as a mechanism to clear myosin aggregates. This multifaceted approach will provide novel insights into the developmental and biophysical bases of MSM and yield potential therapeutic approaches that may be useful for treating MSM and other inclusion body diseases.