Titin Splicing Mechanisms and Physical Implications

说明(申请人提供)：肌动蛋白是一种非常大的蛋白质，主要存在于心肌和骨骼肌中。它是交替剪接的，不同的剪接异构体影响蛋白质的功能。我们已经对大鼠的一种突变进行了部分表征，该突变显著改变了心脏中肌动蛋白的剪接模式。导致肌动蛋白剪接改变的突变基因已经定位于1号染色体上的RNA结合蛋白。将含有RBM20序列的腺病毒载体从纯合子突变株导入培养的心肌细胞将用于拯救实验。将使用芯片分析来探索RBM20与RNA或DNA的结合。将探索Titin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 blotting对RBM20信息和蛋白的发育上调，RBM20磷酸化状态的发育变化，或者与RBM20相关的其他一个或多个蛋白质的发育变化。为了与压力和容量超负荷挑战后的野生型动物进行比较，我们将利用Titin剪接突变模型来探索Titin在拉伸依赖信号转导中的作用。将进行使用杂合子突变体来平衡Titin拉伸信号的实验，以及通过丙基硫氧嘧啶改变Titin表达的模型系统。基因表达的变化将使用定量聚合酶链式反应和蛋白质印迹法来测量。将进行研究，以表征纯合突变的心肌细胞动作电位延长的持续时间，并确定在分离细胞中发现的改变的离子通道属性是否也在体内观察到。我们还将使用突变的大鼠模型，利用分离的皮肤心肌细胞来探索Titin在长度依赖激活和Frank-Starling关系中的作用。用剥皮的骨小梁进行的X射线衍射实验将比较野生型、杂合子和纯合子突变体在不同肌节长度下的纤维间距。概述的研究应该为肌动蛋白的功能和控制其异构体表达的机制提供新的见解。它们还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：已证明在扩张型心肌病中会发生Titin亚型的变化，因此了解控制Titin剪接的机制以及心脏对不同Titin亚型的适应性可能有助于设计这种普遍存在的心血管疾病的治疗方法。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。

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.