Mechanistic basis and potential therapies for myosin storage myopathy

描述（由申请人提供）：我们建议建立和分析首次报道的肌球蛋白储存性肌病（MSM）动物模型，这是一种由慢/ β -心肌肌球蛋白重链c端棒区点突变引起的人类骨骼肌和心肌退行性疾病。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。果蝇系统将允许我们在标准化的遗传背景下检查每个MSM等位基因的纯合子和杂合子（突变/+）。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。我们将验证以下假设：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)在纤维组装中存在缺陷，b)显示异常的纤维降解和/或c)易于聚集，3)阻止MSM肌球蛋白聚集形成或增强MSM肌球蛋白周转可以改善突变肌肉结构和性能。为了验证这些假设，我们将追求以下具体目标：1)研究四种不同的MSM突变在衰老过程中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分析来探讨MSM的进展性，并将我们的结果与现存的人类数据联系起来。2)分离突变型肌球蛋白分子，评估其成丝能力、蛋白水解稳定性和聚集倾向。这将有助于确定这种疾病的分子基础。3)尝试改善生物体的疾病表型a)通过过表达分子伴侣蛋白α -结晶蛋白，Hsp70， Hsp90或UNC-45（所有已知有助于肌球蛋白折叠和/或保护免受压力）；B)利用热休克反应的小分子诱导剂更广泛地诱导分子伴侣的表达；C)利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，并产生潜在的治疗方法，可能对治疗MSM和其他包涵体疾病有用。

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.