Function of titin & a novel nebulin in heart development

尽管在了解主动收缩的分子基础方面取得了重大进展，但令人惊讶的是，人们对心肌细胞中肌原纤维形成的控制机制知之甚少。很可能，确定分别属于“第三和第四灯丝系统”的titin和nebulin的独特分子性质，将为这些未解决的问题提供分子解释。这项研究的长期目标是确定在肌原纤维组装过程中调节收缩蛋白相互作用的分子成分和机制。本提案的目标是通过一个新的心肌肌原纤维组装模型来确定titin、titin结合蛋白T-cap和一种新的心脏nebulin相关蛋白在调节粗纤维和细纤维组装、长度和组织中的分子作用，该模型忠实地概括了肌原纤维在体内的组装。目的：(1)完成心肌细胞分化过程中肌原纤维组装的研究。从禽类胚胎心脏前区提取的外植体将用于研究厚丝蛋白和细丝蛋白的外观和组装的时间模式，以及它们与titin、T-cap和nebulin相关蛋白的关系。(2)克隆和表征编码新型nebulin相关蛋白的cdna，并创建用于研究其功能的分子试剂（用于目标3和目标4）。(3)通过过表达确定的片段来确定titin、T-cap和nebulin相关蛋白的作用，这些片段作为显性阴性抑制剂使用腺病毒递送。此外，还将确定titin， T-cap和nebulin相关蛋白的特定区域，以靶向其组装成肌原纤维。(4)利用反义策略和靶向基因突变检测小鼠胚胎干细胞中肌原纤维形成过程中titin、T-cap和nebulin相关蛋白功能表达的降低作用。建立体外心肌纤维组装模型，确定titin、T-cap和nebulin在调节粗、细纤维组装和动力学中的作用，对于了解包括家族性肥厚性心肌病在内的各种类型心肌病的分子基础至关重要。

Despite major advances in understanding the molecular basis of active contraction, surprisingly little is known about mechanisms controlling myofibril formation in cardiac myocytes. It is likely that determination of the unique molecular properties of titin and nebulin, members of the "third and fourth filament systems" respectively, will provide a molecular explanation of these unsolved questions. The long term objective of this research is to identify the molecular components and mechanisms that regulate contractive protein interactions during myofibril assembly.. The goal of this proposal is to define the molecular role of titin, the titin binding protein T-cap, and a novel cardiac nebulin-related protein in the regulation of thick and thin filament assembly, length and organization using a model of de novo cardiac myofibril assembly that faithfully recapitulates myofibril assembly in vivo. The Aims are: (1) To complete the investigation of myofibril assembly in differentiation cardiac myocytes. Explants from pre-cardiac regions of avian embryos will be used to examine the temporal pattern of appearance and assembly of thick and thin filament proteins, and their relationship to titin, T-cap and nebulin-related protein. (2) To clone and characterize cDNAs encoding a novel nebulin-related protein, and to create molecular reagents (for aims 3 and 4) for investigating its function(s). (3) To determine the role(s) of titin, T-cap and the nebulin-related protein by over-expression of defined fragments that act as dominant negative inhibitors using adenoviral delivery. Also, specific regions of titin, T-cap and nebulin-related protein required for targeting their assembly into myofibrils will be identified. (4) To examine the effects of reducing functional expression of titin, T-cap and nebulin-related protein during myofibrillogenesis using antisense strategies and by targeting gene mutation in mouse embryonic stem cells. Establishment of an in vitro model for cardiac myofibril assembly and identifying the roles of titin, T-cap and nebulin of modulating thick and thin filament assembly and dynamics are pivotal for understanding the molecular bases of various types of cardiac myopathies, including familial hypertrophic cardiomyopathies.