Function of titin & a novel nebulin in heart development

尽管对主动收缩的分子基础的理解取得了重大进展，但令人惊讶的是，对心肌细胞中肌原纤维形成的控制机制知之甚少。很可能，确定的独特分子特性的titin和nebulin，成员的“第三和第四丝系统”，将提供一个分子解释这些未解决的问题。本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组分和机制。该提案的目标是定义肌联蛋白的分子作用，肌联蛋白结合蛋白T-cap，和一种新的心脏星云蛋白相关的蛋白质在调节粗细丝组装，长度和组织使用的模型从头心脏肌原纤维组装，忠实地重演体内肌原纤维组装。目的：（1）完成分化心肌细胞肌原纤维组装的研究。从禽胚胎的心脏前区域的外植体将被用来检查的外观和组装的粗和细丝蛋白的时间模式，以及它们的关系，肌联蛋白，T-cap和星云蛋白相关蛋白。(2)克隆和鉴定编码一种新的星云蛋白相关蛋白的cDNA，并创建用于研究其功能的分子试剂（目标3和4）。(3)通过使用腺病毒递送作为显性负抑制剂的确定片段的过表达来确定肌联蛋白、T-cap和星云蛋白相关蛋白的作用。此外，肌联蛋白，T-cap和nebulin相关蛋白的特定区域所需的靶向其组装成肌原纤维将被确定。(4)目的：研究肌联蛋白（titin）、T-cap和nebulin相关蛋白在肌原纤维形成过程中的功能性表达，并通过反义策略和靶向基因突变对小鼠胚胎干细胞的影响。建立心肌原纤维组装的体外模型，明确肌联蛋白、T-cap和nebulin在调节粗、细肌丝组装和动力学中的作用，对于理解包括家族性肥厚性心肌病在内的各种心肌病的分子基础具有重要意义。

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.