Role of troponin T isoforms in nemaline myopathy

肌钙蛋白 T 同工型在线状肌病中的作用

• 批准号: 7743295
• 负责人: Jian-Ping Jin
• 金额: $ 34.2万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7743295
• 关键词: 3 year old; Acidosis; Adult; Affect; Age; Alleles; Amino Acids; Amish; Animal Model; Apoptosis; Appearance; Arts; Basic Science; Binding; Biochemical; Biological; Biology; Birth; Breast; Breast Diseases; Cardiac; Case Study; Cells; Cessation of life; Characteristics; Charge; Chickens; Child; Clinic; Clinical Medicine; Codon Nucleotides; Communities; Complement; Complex; Contracture; County; Cytosol; Deformity; Development; Disease; Disease Progression; Dissociation; Dominant-Negative Mutation; Down-Regulation; Embryo; Enhancers; European; Event; Exhibits; Family; Fatigue; Fiber; Figs - dietary; Financial compensation; Frequencies; Functional disorder; Future; Gene Dosage; Gene Expression; General Population; Genes; Genetic; Goals; Haploidy; Healthcare; Hereditary Disease; Heterozygote; Immigrant; Incidence; Individual; Infant; Inherited; Investigation; Isoelectric Point; Knock-in Mouse; Life; Link; Living Wills; Medicine; Messenger RNA; Metabolic; Methods; Microfilaments; Molecular; Molecular Genetics; Muscle; Muscle Cells; Muscle Contraction; Muscle Development; Muscle Fibers; Muscle Tremors; Muscle Weakness; Muscle function; Muscle hypotonia; Muscular Atrophy; Mutagenesis; Mutant Strains Mice; Mutation; Myofibrils; Myopathy; N-terminal; Names; Nemaline Myopathies; Neonatal; Neuromuscular Diseases; Nonsense Mutation; Nuclear Family; Nucleotides; Ohio; Pathogenesis; Pathology; Patients; Pennsylvania; Perinatal; Phenotype; Physiological; Play; Population; Postpartum Period; Progress Reports; Protein Isoforms; Proteins; Proteolysis; RNA Splicing; Rare Diseases; Reagent; Regulation; Reporting; Research; Research Personnel; Research Project Grants; Respiratory Failure; Respiratory Insufficiency; Role; Shapes; Skeletal Muscle; Slow-Twitch Muscle Fibers; Stress; Striated Muscles; Supplementation; Symptoms; System; Termination of pregnancy; Terminator Codon; Testing; Therapeutic; Thin Filament; Time; Tremor; Tropomyosin; Troponin; Troponin T; United States National Institutes of Health; Up-Regulation; Vertebrates; Work; animal tissue; base; bench to bedside; career; cytotoxicity; dosage; effective therapy; fetal; fetal diagnosis; genetic regulatory protein; in utero; insight; loss of function; meetings; mouse model; muscle degeneration; novel; polypeptide; postnatal; promoter; public health relevance; retinal rods; skeletal; wasting

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.

描述（由申请人提供）：Amish Nemaline Myopathy （ANM）是一种常染色体隐性肌肉疾病，在宾夕法尼亚州和俄亥俄州的Old Order Amish人中发现，每~500个新生儿中就有1个患病。ANM等位基因包含慢骨骼肌肌钙蛋白T （TnT）基因（TNNT1）的无义突变，导致TnT蛋白在氨基酸179处截断。这种截断的慢速TnT不能并入肌原纤维，并在肌细胞内降解。从表型上看，ANM患者会出现肌肉震颤、挛缩和肌张力过低。受影响的婴儿在出生时在临床上是正常的，但迅速发展为通常在第二年因呼吸衰竭而导致死亡的ANM症状。目前尚无有效的治疗方法。本研究项目旨在了解ANM的发病机制和ANM肌肉的病理生理，以达到治疗ANM的最佳目标。提出以下三个具体目的：目的一：表征慢速TnT缺乏的动物模型。我们将在慢速TnT敲除小鼠模型中研究骨骼肌功能，在该模型中，慢速TnT基因表达降低导致慢速TnT蛋白减少、肌肉萎缩和向快纤维表型转换，同时对疲劳的耐受性降低。我们还将研究在慢速TnT基因中敲除ANM无义突变的小鼠模型的表型，以研究其发病机制和疾病进展。目的二：探讨慢速TnT基因单倍体和截短慢速TnT的细胞毒性对ANM发病机制和病理生理的条件作用。ANM杂合子报告了间接的肌肉症状。我们将研究半剂量的慢速TnT基因对杂合子慢速TnT敲入突变小鼠肌肉功能的潜在影响。非肌丝相关的TnT碎片表现出细胞毒性。我们将研究ANM慢速TnT片段的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：研究心脏骨骼肌中TnT的调节和功能，为ANM患者制定治疗代偿方案提供依据。我们已经在ANM亚型中发现证据，ANM中缓慢的骨骼TnT功能的丧失可能部分由骨骼肌中心脏TnT的持续表达来补偿。这种代偿性心脏TnT基因表达的功能意义和激活将在蛋白质、肌肉细胞和组织以及动物水平上进行研究，以开发针对ANM的特异性治疗方法。在另一种方法中，也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和新的实验系统。这一结果将极大地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肉无力和骨骼肌纤维杆状“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肌病都是由肉瘤细丝蛋白突变引起的。Amish Nemaline Myopathy （ANM）是一种致命性的遗传性Nemaline Myopathy，在宾夕法尼亚州和俄亥俄州的Old Order Amish社区发病率非常高（1 / 500）。ANM的遗传基础是编码肌钙蛋白T （TnT）的慢骨骼肌同种异构体的基因的单核苷酸无义突变，肌钙蛋白T是一种肌肉特异性Ca2+调节蛋白。这种无义突变截断了氨基酸179处的慢速TnT多肽，使TnT蛋白无法与肌原纤维结合并迅速降解。这种功能丧失的机制与该病的常染色体隐性遗传一致。从表型上看，ANM患者会出现肌肉震颤、挛缩和肌张力过低。出生时症状微不足道，但迅速恶化，通常在第二年因呼吸衰竭而死亡。目前尚无有效的治疗方法。虽然ANM在一般人群中是一种罕见的疾病，但其统一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。我们是目前世界上唯一研究ANM的研究团队，迄今为止我们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。本研究项目旨在通过研究TnT异构体的功能和调控，进一步推动我们在治疗方面的进展。我们将研究缓慢的tnt缺乏动物模型，以了解ANM病理和肌肉功能。我们将研究慢速TnT基因剂量减少对肌肉功能的潜在影响，以及截断的慢速TnT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，我们将重点研究在缓慢的TnT缺乏骨骼肌中代偿性心脏TnT表达的功能意义和激活，以作为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，我们也将对不同的TnT亚型和Ca2+调控横纹肌收缩的功能意义有重要的认识，这是生物学和医学的一个基础课题。