Function of titin & a novel nebulin in heart development

尽管在理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，人们对心肌细胞肌原纤维形成的控制机制知之甚少。肌动蛋白和星云蛋白分别属于“第三丝系统”和“第四丝系统”，它们独特的分子性质的确定很可能为这些悬而未决的问题提供一个分子解释。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组成和机制。这项建议的目的是利用一种新的心肌肌原纤维组装模型来确定Titin、Titin结合蛋白T-Cap和一种新的心脏星云蛋白相关蛋白在调节粗丝和细丝组装、长度和组织中的分子作用。其目的是：(1)完成肌原纤维在分化心肌细胞中组装的研究。来自禽类胚胎心前区的外植体将被用来研究粗丝和细丝蛋白的出现和组装的时间模式，以及它们与肌动蛋白、T-帽和星云蛋白相关蛋白的关系。(2)克隆和鉴定一种新的星云蛋白相关蛋白的编码基因，并建立用于研究其功能的分子试剂(AIMS 3和4)(S)。(3)通过腺病毒载体过度表达作为显性负性抑制因子的特定片段，确定肌动蛋白、T-CAP和星云蛋白相关蛋白的作用(S)。此外，还将确定将其组装成肌原纤维所需的肌蛋白、T-帽和星云蛋白相关蛋白的特定区域。(4)应用反义策略和靶向基因突变的方法，研究在小鼠胚胎干细胞肌原纤维形成过程中抑制肌动蛋白、T-帽和烟雾素相关蛋白功能表达的作用。建立心肌肌原纤维组装的体外模型，确定肌蛋白、T-帽和星云蛋白在调控粗丝和细丝组装和动力学中的作用，对于理解包括家族性肥厚型心肌病在内的各种类型心肌疾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.