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Titin Splicing Mechanisms and Physical Implications

肌联蛋白剪接机制和物理意义

基本信息

批准号：
7851384
负责人：
MARION Lewis GREASER
金额：
$ 36.31万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7851384
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

项目摘要

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）：Titin是一种主要存在于心脏和骨骼肌中的巨大蛋白质。它是选择性剪接的，不同的剪接异构体影响蛋白质的功能。我们已经部分描述了老鼠体内的一种突变，这种突变极大地改变了心脏中titin的剪接模式。负责改变titin剪接的突变基因已定位于1号染色体上的RNA结合蛋白。将含有Rbm20序列的腺病毒构建体转染到纯合突变体培养的心肌细胞中，将用于抢救实验。Rbm20与RNA或DNA的结合将通过ChIP试验进行探索。其他机制，其中titin剪接是发展改变将探讨。这些包括分别使用定量PCR和Western blotting对Rbm20信息和蛋白质的发育上调，Rbm20磷酸化状态的发育改变，或与Rbm20相关的其他蛋白质或蛋白质的发育变化。将利用titin剪接突变模型与野生型动物在压力和体积过载挑战后的比较，探讨titin在拉伸依赖性信号转导中的作用。使用杂合子突变体和丙硫脲改变titin表达的模型系统来平衡titin拉伸信号的实验将被进行。使用定量PCR和Western blots检测基因表达的变化。将进行研究，以表征纯合突变体心肌细胞动作电位持续时间的延长，并确定在分离细胞中发现的离子通道特性的改变是否也在体内观察到。我们还将使用突变大鼠模型来探索titin在长度依赖性激活中的作用以及使用分离的皮肤心肌细胞的Frank-Starling关系。利用剥皮小梁进行X射线衍射实验，比较野生型、杂合子和纯合子突变体在不同肌节长度下的丝间距。概述的研究应该为titin的功能和控制其异构体表达的机制提供新的见解。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：已证实在扩张型心肌病中会发生Titin亚型的改变，因此了解控制Titin剪接的机制以及心脏对不同Titin亚型的适应可能有助于设计治疗这种常见心血管疾病的方法。此外，我们建立的大鼠突变模型应该有助于理解心律失常和猝死的机制。