Titin Splicing Mechanisms and Physical Implications

肌联蛋白剪接机制和物理意义

• 批准号: 7655665
• 负责人: MARION Lewis GREASER
• 金额: $ 41万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7655665
• 关键词: Action Potentials; Adenoviruses; Adult; Affect; Alternative Splicing; Animals; Antibodies; Arrhythmia; Binding; Biological Assay; Biological Models; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular system; Cell Nucleus; Cells; Chromosomes, Human, Pair 1; Confocal Microscopy; Cytoplasm; DNA; Data; Development; Dilated Cardiomyopathy; Down-Regulation; Electrocardiogram; Equilibrium; Filament; Gene Expression; Genes; Genotype; Health; Heart; Heart Atrium; Heterozygote; Homozygote; Human; Ion Channel; Ions; Length; Measures; Mechanics; Mediating; Messenger RNA; Modeling; Muscle Cells; Mutate; Mutation; Myofibrils; Pattern; Phenotype; Phosphorylation; Play; Property; Protein Isoforms; Proteins; Protocols documentation; RNA; RNA Splicing; RNA-Binding Proteins; Rattus; Regulation; Relative (related person); Role; Sarcomeres; Signal Transduction; Skeletal Muscle; Skin; Staining method; Stains; Stretching; Sturnus vulgaris; Sudden Death; Testing; Time; Transfection; Trypsin; Up-Regulation; Ventricular; Western Blotting; Work; X ray diffraction analysis; X-Ray Diffraction; cell behavior; connectin; heart function; in vivo; insight; mutant; novel; pressure; protein distribution; protein function; public health relevance; research study; response; tool

DESCRIPTION (provided by applicant): Titin is an extremely large protein found primarily in cardiac and skeletal muscle. It is alternatively spliced and different splice isoforms affect the function of the protein. We have partially characterized a mutation in rats that dramatically alters the splicing pattern of titin in the heart. The mutated gene responsible for altered titin splicing has been localized to an RNA binding protein in chromosome 1. Transfection of adenovirus constructs contain the Rbm20 sequence into cultured cardiomyocytes from homozygous mutants will be used in rescue experiments. Binding of the Rbm20 to RNA or DNA will be explored using ChIP assays. Alternative mechanisms whereby titin splicing is developmentally changed will be explored. These include developmental up regulation of Rbm20 message and protein using quantitative PCR and Western blotting respectively, developmental alterations in Rbm20 phosphorylation state, or developmental changes in other protein or proteins that associate with Rbm20. The role of titin in stretch dependent signal transduction will be explored using the titin splice mutation model for comparisons with wild type animals after pressure and volume overload challenges. Experiments where the titin stretch signals can be balance using heterozygote mutants and a model system where titin expression is altered by propylthiouricil will be conducted. Changes in gene expression will be measured using quantitative PCR and Western blots. Studies will be conducted to characterize the prolonged duration of action potentials from cardiomyocytes of homozygous mutants and determine whether the altered ion channel properties found in isolated cells are also observed in vivo. We will also use the mutant rat model to explore titin's role in length dependent activation and the Frank-Starling relationship using isolated skinned cardiomyocytes. X- ray diffraction experiments using skinned trabeculae will compare filament spacing of wild type, heterozygote, and homozygote mutants at different sarcomere lengths. The studies outlined should provide new insights into titin function and the mechanisms controlling its isoform expression. They may also help to better understand the role of ion channel alternative splicing on cardiac arrhythmias. PUBLIC HEALTH RELEVANCE: Titin isoforms changes have been shown to occur in dilated cardiomyopathy, so understanding the mechanisms controlling titin splicing and the cardiac adaptations to different titin isoforms may help in devising treatments for this prevalent cardiovascular disease. In addition the rat mutation model we have developed should help to understand mechanisms of cardiac arrhythmia and sudden death.

描述（由申请人提供）：肌联蛋白是一种主要在心肌和骨骼肌中发现的极大蛋白质。它是可变剪接的，不同的剪接异构体影响蛋白质的功能。我们已经部分地表征了大鼠中的突变，该突变极大地改变了心脏中肌联蛋白的剪接模式。负责改变肌联蛋白剪接的突变基因已定位于1号染色体中的RNA结合蛋白。将含有Rbm 20序列的腺病毒构建体转染到来自纯合突变体的培养的心肌细胞中将用于拯救实验。将使用ChIP测定探索Rbm 20与RNA或DNA的结合。将探讨肌联蛋白剪接在发育过程中发生变化的替代机制。这些包括分别使用定量PCR和Western印迹法的Rbm 20信息和蛋白质的发育上调，Rbm 20磷酸化状态的发育改变，或与Rbm 20相关的其他蛋白质或蛋白质的发育变化。将使用肌联蛋白剪接突变模型探索肌联蛋白在拉伸依赖性信号转导中的作用，以在压力和体积超负荷挑战后与野生型动物进行比较。将进行使用杂合子突变体和其中肌联蛋白表达被丙基硫氧嘧啶改变的模型系统来平衡肌联蛋白拉伸信号的实验。将使用定量PCR和蛋白质印迹法测量基因表达的变化。将进行研究以表征纯合突变体心肌细胞的动作电位持续时间延长，并确定是否也在体内观察到分离细胞中发现的改变的离子通道特性。我们也将使用突变的大鼠模型，探讨肌联蛋白的作用，长度依赖性激活和Frank-Starling关系，使用分离的皮肤心肌细胞。使用去皮小梁的X射线衍射实验将比较不同肌节长度的野生型、杂合子和纯合子突变体的肌丝间距。概述的研究应提供新的见解肌联蛋白的功能和控制其异构体表达的机制。它们也可能有助于更好地理解离子通道选择性剪接在心律失常中的作用。公共卫生相关性：肌联蛋白异构体的变化已被证明发生在扩张型心肌病，因此了解控制肌联蛋白剪接和心脏适应不同肌联蛋白异构体的机制可能有助于设计治疗这种流行的心血管疾病。此外，我们开发的大鼠突变模型将有助于了解心律失常和猝死的机制。