Modeling Inheritable Myofibrillar Myopathy with Patient-Specific iPS Cells

使用患者特异性 iPS 细胞模拟遗传性肌原纤维肌病

• 批准号: 9196262
• 负责人: KATIE ANN MITZELFELT
• 金额: $ 1.12万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-08 至 2016-11-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9196262
• 关键词: Accounting; Actins; Affect; Age; Animal Model; Biological Models; Biopsy; Cardiomyopathies; Cataract; Cell Cycle; Cell Cycle Progression; Cell Line; Cell model; Cells; Crystallins; DNA Sequence Alteration; Data; Dermal; Desmin; Development; Disease; Disease model; Exhibits; Fibroblasts; Future; Gene Expression; Gene Proteins; Genes; Genetic; Grant; Heat shock proteins; Human; In Vitro; Injury; Intermediate Filament Proteins; Knockout Mice; Life; Literature; Maintenance; Modeling; Molecular Chaperones; Molecular Weight; Mus; Muscle; Muscle Contraction; Muscle Fibers; Muscle Weakness; Muscular Atrophy; Mutation; Myocardium; Myopathy; Natural regeneration; Nonsense Mutation; Pathology; Patients; Phenotype; Preclinical Drug Evaluation; Process; Protocols documentation; Research; Respiration; Respiratory Diaphragm; Role; Sarcomeres; Severities; Skeletal Muscle; Specificity; Staging; Staining method; Stains; Stem cells; Stress; Structure; Symptoms; System; Tissues; Ursidae Family; cell type; connectin; design; gain of function; gene correction; genetic manipulation; induced pluripotent stem cell; infancy; insight; loss of function; muscle degeneration; muscle regeneration; muscle stiffness; mutant; myogenesis; overexpression; progenitor; protein aggregate; protein expression; public health relevance; satellite cell; self-renewal; skeletal; skeletal muscle differentiation; transcription factor

 DESCRIPTION (provided by applicant): Multiple mutations in the gene encoding aB-crystallin, CRYAB, have been identified in the literatures that result in skeletal myopathy. αB-Crystallin is small molecular-weight heat shock protein that has been shown to function as a chaperone for desmin, titin, and actin. Additionally, αB-crystallin impacts skeletal muscle differentiation throgh exhibiting effects on cell cycle exit and a muscle-specific, regulatory transcription factor, MyoD. A patient harboring the homozygous recessive, nonsense mutation, 343delT, in CRYAB develops symptoms of severe muscle stiffness and degeneration around age four months, following normal development. The patient's muscle biopsy shows dense, irregular staining of 343delT, possibly reminiscent of the aggregates visible with other mutant forms of aB-crystallin, such as R120G, which has been shown to also induce the aggregation of an intermediate filament protein, desmin, contributing to the disease pathology. The patient's symptoms also bear similarities to the CRYAB/HSPB2 double knockout mouse, though the mouse seems to have less severe and later onset muscle deterioration. Therefore, the question as to whether this disease pathology results from loss of αB-crystallin function or gain of toxic function of 343delT αB-crystallin remains and whether this impact is seen in myogenic progenitor cells, differentiated myotubes, or both. This proposal aims to examine this question using 343delT patient-specific induced pluripotent stem cell- (iPSC) derived myogenic progenitors and skeletal myotubes. iPSCs offer great utility in modeling cell autonomous diseases in vitro because of their continuous ability to self-renew and pluripotent differentiation potential; they are also amenable to genetic manipulation. iPSCs will be differentiated to myogenic progenitors and skeletal myotubes by an existing protocol. [Control iPSCs have been generated by gene correction in 343delT patient iPSCs] and will be used for phenotypic comparisons to examine the structure and function of differentiated myotubes as well as analyze cell cycle progression and gene expression throughout differentiation of myogenic progenitors to myotubes. Additionally, CRYAB null iPSCs have been generated from 343delT patient iPSCs for comparison to determine if phenotypes are due to loss or gain of function effects of the 343delT mutation. Successful completion of these aims will provide a human model system of 343delT-induced myopathy and a better understanding of how expression of the mutant and/or lack of expression contribute to myopathy, as well as the importance of a chaperone, aB-crystallin, in human skeletal muscle differentiation and maintenance.

 描述(申请人提供)：文献中已鉴定出编码AB-晶体蛋白基因CryAB的多种突变可导致骨骼肌病。αB-晶体蛋白是一种小分子热休克蛋白，已被证明是结蛋白、肌动蛋白和肌动蛋白的伴侣。此外，αB-晶体蛋白通过影响细胞周期退出和肌肉特异的调节转录因子MyoD来影响骨骼肌的分化。 一名在CryAB中携带343delT纯合子隐性无义突变的患者，在正常发育后，在四个月左右出现严重的肌肉僵硬和退化症状。患者的肌肉活检显示343delT浓密、不规则的染色，可能使人想起与其他AB-晶状体蛋白突变形式(如R120G)一起可见的聚集物，R120G已被证明也可诱导中间丝蛋白结蛋白的聚集，从而导致疾病病理。患者的症状也与CryAB/HSPB2双基因敲除小鼠相似，尽管小鼠的肌肉退化似乎不那么严重，而且发生得更晚。因此，关于本病的病理原因是αB-晶体蛋白功能丧失还是343delTαB-晶体蛋白毒性功能增强，以及这种影响是否见于成肌祖细胞、分化的肌管或两者兼而有之的问题仍然存在。这项建议旨在使用343delT患者特异性诱导多能干细胞(IPSC)来源的肌源性祖细胞和骨骼肌管来研究这一问题。由于IPSCs具有持续的自我更新能力和多潜能分化潜能，在体外模拟细胞自主性疾病方面具有很大的实用价值；它们也容易受到遗传操作的影响。根据现有的方案，IPSCs将分化为肌源性祖细胞和骨骼肌管。[对照IPSCs已在343delT患者IPSCs中通过基因校正产生]，并将用于表型比较，以检查分化的肌管的结构和功能，以及分析肌祖细胞向肌管分化的细胞周期进程和基因表达。此外，从343delT患者的IPSCs中产生了CryAB缺失的IPSCs用于比较，以确定表型是由于343delT突变的功能效应的丧失还是获得。这些目标的成功完成将提供343delT诱导的肌病的人体模型系统，并更好地理解突变和/或表达缺失如何导致肌病，以及伴随蛋白AB-晶体蛋白在人类骨骼肌分化和维持中的重要性。

项目成果

The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility.

与早发性骨骼肌病相关的人类 343delT HSPB5 伴侣会导致蛋白质溶解度缺陷。

• DOI: 10.1074/jbc.m116.730481
• 发表时间: 2016
• 期刊: The Journal of biological chemistry
• 作者: Mitzelfelt,KatieA;Limphong,Pattraranee;Choi,MelindaJ;Kondrat,FrancesDL;Lai,Shuping;Kolander,KurtD;Kwok,Wai-Meng;Dai,Qiang;Grzybowski,MichaelN;Zhang,Huali;Taylor,GraydonM;Lui,Qiang;Thao,MaiT;Hudson,JudithA;Barresi,Rita
• 通讯作者: Barresi,Rita