Role of troponin T isoforms in nemaline myopathy

描述（由申请方提供）：阿米什人线状肌病（ANM）是一种常染色体隐性肌肉疾病，见于宾夕法尼亚州和俄亥俄州的旧秩序阿米什人，每500例新生儿中有1例受影响。ANM等位基因在缓慢骨骼肌肌钙蛋白T（TnT）基因（TNNT 1）中含有无义突变，导致TnT蛋白在氨基酸179处截短。这种截短的慢TnT不能掺入肌原纤维中，并在肌细胞内降解。在表型上，患有ANM的个体遭受肌肉震颤、挛缩和张力减退。受影响的婴儿在出生时临床正常，但迅速发展ANM症状，通常导致第二年因呼吸衰竭而死亡。没有有效的治疗方法。本研究项目旨在了解ANM的发病机制和ANM肌肉的病理生理学，以达到治疗该病的最佳目标。提出了以下三个具体目标：目标I：表征慢TnT缺陷的动物模型。我们将在慢TnT基因敲低小鼠模型中研究骨骼肌功能，其中慢TnT基因表达降低导致慢TnT蛋白降低、肌肉萎缩和向快纤维表型的转变以及对疲劳的耐受性降低。我们还将研究小鼠模型的表型，其中ANM无义突变敲除慢TnT基因，以研究发病机制和疾病进展。目标二：研究慢肌钙蛋白T基因单倍性和截短的慢肌钙蛋白T细胞毒性对ANM发病机制和病理生理学的条件性影响。ANM杂合子报告间接肌肉症状。我们将研究半剂量慢肌钙蛋白T基因对杂合子慢肌钙蛋白T基因敲入突变小鼠肌肉功能的潜在影响。非肌丝相关TnT片段表现出细胞毒性。我们将研究ANM慢TnT片段的这种潜在细胞毒性是否以及如何导致ANM患者的肌肉变性。目的三：研究肌钙蛋白T（TnT）在骨骼肌中的调节和功能，为ANM患者的代偿治疗提供理论依据。我们在ANM的一个亚型中发现了证据，证明ANM中缓慢骨骼肌TnT功能的丧失可能通过骨骼肌中心脏TnT的持续表达而部分补偿。将在蛋白质、肌肉细胞和组织以及动物水平研究这种代偿性心脏TnT基因表达的功能意义和激活，以开发ANM的特异性治疗方法。在替代方法中，还将使用新兴试剂探索ANM无义终止密码子的抑制。基于我们迄今为止所取得的进展，这些研究采用了最先进的分子遗传学、生物化学、细胞生物学和生理学方法以及新颖的实验系统。这些结果将大大促进对这种毁灭性疾病的有效治疗的追求。公共卫生相关性：线状肌病是一组以肌无力和骨骼肌纤维中的杆状“线状”内含物为特征的神经肌肉疾病。所有已知的遗传性线状体肌病都是由肌节细丝蛋白突变引起的。阿米什人线状体肌病（ANM）是一种致命的遗传性线状体肌病，在宾夕法尼亚州和俄亥俄州的旧秩序阿米什人社区中发病率非常高（每500名新生儿中有1名）。ANM的遗传基础是编码肌钙蛋白T（TnT）的慢骨骼肌亚型（一种肌肉特异性钙调节蛋白）的基因中的单核苷酸无义突变。这种无义突变在氨基酸179处截短慢TnT多肽，并使TnT蛋白不能掺入肌原纤维中并被快速降解。这种功能丧失机制与该病的常染色体隐性遗传一致。在表型上，患有ANM的个体遭受肌肉震颤、挛缩和张力减退。出生时症状轻微，但迅速恶化，通常导致第二年呼吸衰竭死亡。目前没有有效的治疗方法。虽然ANM在一般人群中是一种罕见的疾病，但其一致的破坏性进展与已经阐明的分子原因相结合，值得进一步研究。在宾夕法尼亚州和俄亥俄州，每年有两到三个ANM婴儿出生，没有一个能活到3岁。我们目前是世界上唯一一个致力于ANM的研究团队，我们迄今为止的进展已经给受影响的家庭和一般阿米什社区带来了在不久的将来获得有效治疗的希望。该研究项目旨在通过研究TnT亚型的功能和调节来进一步开发治疗方法。我们将研究慢性TnT缺陷动物模型，以了解ANM病理和肌肉功能。我们将研究降低慢肌钙蛋白T基因剂量对肌肉功能的潜在影响和截短的慢肌钙蛋白T的细胞毒性与ANM肌肉变性的联系。在探索抑制ANM无义终止密码子的可能性的同时，我们将专注于研究补偿性心脏TnT表达在缓慢TnT缺陷骨骼肌中的功能意义和激活，以用作ANM的特异性治疗。ANM是唯一已知的由TnT基因隐性突变引起的疾病。通过研究ANM的病理生理学，我们还将获得重要的见解不同TnT亚型的功能意义和横纹肌收缩的Ca2+调节，这是生物学和医学的一个基本课题。

DESCRIPTION (provided by applicant): Amish Nemaline Myopathy (ANM) is an autosomal recessive muscle disorder found among the Old Order Amish in Pennsylvania and Ohio, affecting 1 out of every ~500 births. The ANM allele contains a nonsense mutation in the slow skeletal muscle troponin T (TnT) gene (TNNT1), which results in truncation of the TnT protein at amino acid 179. This truncated slow TnT cannot incorporate into myofibrils and is degraded inside the myocyte. Phenotypically, individuals with ANM suffer from muscle tremors, contractures and hypotonia. The affected infants are clinically normal at birth but rapidly develop the ANM symptoms that usually result in death from respiratory failure during the second year. No effective treatment is available. This research project aims to understanding the pathogenesis of ANM and the pathophysiology of ANM muscle for the optimal goal of developing a cure of the disease. The following three specific aims are proposed: Aim I: To characterize animal models deficient in slow TnT. We shall study the skeletal muscle function in a slow TnT knockdown mouse model in which lowered slow TnT gene expression results in decreased slow TnT protein, muscle atrophy and a switch to fast fiber phenotypes with decreased tolerance to fatigue. We shall also study the phenotype of a mouse model in which the ANM nonsense mutation is knocked in the slow TnT gene to investigate the pathogenesis and disease progression. Aim II: To examine the conditional effects of slow TnT gene haploidy and cytotoxicity of the truncated slow TnT on ANM pathogenesis and pathophysiology. ANM heterozygotes have reported circumstantial muscle symptoms. We shall investigate the potential effect of half dosage of slow TnT gene on muscle function in heterozygote slow TnT knock-in mutant mice. Non-myofilament-associated TnT fragments exhibit cytotoxicity. We shall examine whether and how this potential cytotoxicity of the ANM slow TnT fragment contributes to muscle degeneration in ANM patients. Aim III: To study the regulation and function of cardiac TnT in skeletal muscle in order to develop therapeutic compensation in ANM patients. We have found evidence in a subtype of ANM that the loss of slow skeletal TnT function in ANM may be partially compensated for by the continuing expression of cardiac TnT in skeletal muscles. Functional significance and activation of this compensatory cardiac TnT gene expression will be investigated at protein, muscle cell and tissue, and animal levels toward the development of a specific treatment for ANM. In an alternative approach, suppression of the ANM nonsense stop codon will also be explored using emerging reagents. Based on progresses we have made to date, these studies employ state-of-the-art molecular genetic, biochemical, cell biological and physiological methods and novel experimental systems. The results will significantly further the pursuit of an effective therapy for this devastating disease. PUBLIC HEALTH RELEVANCE: Nemaline myopathies are a group of neuromuscular disorders characterized by muscle weakness and rod-shaped "nemaline" inclusions in skeletal muscle fibers. All known inherited nemaline myopathies are caused by mutations in sarcomeric thin filament proteins. Amish Nemaline Myopathy (ANM) is a lethal inherited nemaline myopathy present at very high incidences (1 out of 500 births) in the Old Order Amish communities in Pennsylvania and Ohio. The genetic basis of ANM is a single nucleotide nonsense mutation in the gene encoding the slow skeletal muscle isoform of troponin T (TnT), a muscle-specific Ca2+- regulatory protein. This nonsense mutation truncates the slow TnT polypeptide at amino acid 179 and renders the TnT protein incapable of incorporating into myofibrils and being rapidly degraded. This loss of function mechanism is consistent with the autosomal recessive inheritance of the disease. Phenotypically, individuals with ANM suffer from muscle tremors, contractures and hypotonia. Symptoms are trivial at birth but rapidly worsen, usually resulting in death from respiratory failure during the second year. No effective treatment is currently available. Although ANM is a rare disease in the general population, its uniformly devastating progression combined with an already elucidated molecular cause merits much further investigation. Two to three ANM babies are born every year in Pennsylvania and Ohio; none will live to be 3 years old. We are currently the only research team in the world working on ANM and our progress thus far has already given the affected families and the general Amish community a hope for effective treatments in the near future. This research project aims to further our progress towards the development of a therapy by investigating TnT isoform function and regulation. We will study slow TnT-deficient animal models for the understanding of ANM pathology and muscle function. We will examine the potential effect of decreased slow TnT gene dosage on muscle function and the cytotoxicity of truncated slow TnT for links to muscle degeneration in ANM. Together with exploring the possibility of suppressing the ANM nonsense stop codon, we will focus on investigating the functional significance and activation of compensatory cardiac TnT expression in slow TnT-deficient skeletal muscle for use as a specific treatment for ANM. ANM is the only known disease caused by a recessive mutation in a TnT gene. By investigating the pathophysiology of ANM, we will also gain important insights into the functional significance of different TnT isoforms and the Ca2+-regulation of striated muscle contraction, a fundamental topic in biology and medicine.