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（由申请人提供）：文献中已鉴定出编码αB-晶状体蛋白CRYAB的基因中的多个突变会导致骨骼肌病。αB-晶状体蛋白是一种小分子量热休克蛋白，已被证明具有作为骨骼肌病的功能。此外，αB-晶状体蛋白通过对细胞周期退出和肌动蛋白的影响来影响骨骼肌分化。肌肉特异性调节转录因子，MyoD。 CRYAB 中携带纯合隐性无义突变 343delT 的患者在正常发育后约四个月大时出现严重肌肉僵硬和退化的症状。患者的肌肉活检显示 343delT 的致密、不规则染色，可能不存在可见的聚集体。 aB-晶状体蛋白的其他突变形式，例如 R120G，已被证明也能诱导中间丝蛋白的聚集，该患者的症状也与 CRYAB/HSPB2 双敲除小鼠有相似之处，尽管该小鼠的肌肉退化似乎较轻且较晚发生，因此，该疾病的病理学是否是由丧失引起的问题。 αB-晶状体蛋白功能的丧失或 343delT αB-晶状体蛋白的毒性功能的获得仍然存在，以及这种影响是否出现在肌源性祖细胞、分化的肌管或两者中。使用 343delT 患者特异性诱导多能干细胞 (iPSC) 衍生的肌源性祖细胞和骨骼肌管来研究这个问题，因为它们具有持续的自我更新能力和多能分化潜力，因此在体外模拟细胞自主疾病方面具有很大的用途；也可以通过现有的方案将 iPSC 分化为肌源性祖细胞和骨骼肌管。 iPSC 是通过 343delT 患者 iPSC 中的基因校正生成的，将用于表型比较，以检查分化肌管的结构和功能，并分析整个肌管中肌源性祖细胞的细胞周期进展和基因表达分化。此外，CRYAB 无效 iPSC。已从 343delT 患者 iPSC 中生成，用于比较以确定表型是否是由于 343delT 功能效应的丧失或增强所致成功完成这些目标将提供 343delT 诱导的肌病的人类模型系统，并更好地了解突变体的表达和/或表达缺乏如何导致肌病，以及伴侣 aB-晶状体蛋白的重要性。 ，在人体骨骼肌的分化和维持中。

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。

• 发表时间: 2016
• 作者: Mitzelfelt, Katie A;Limphong, Pattraranee;Choi, Melinda J;Kondrat, Frances D L;Lai, Shuping;Kolander, Kurt D;Kwok, Wai;Dai, Qiang;Grzybowski, Michael N;Zhang, Huali;Taylor, Graydon M;Lui, Qiang;Thao, Mai T;Hudson, Judith A;Barresi, Rita
• 通讯作者: Barresi, Rita