ALTERED MECHANICAL LOADS AND SKELETAL MUSCLE PHENOTYPE

肌肉生物学中一个主要的悬而未决的问题涉及确定机械负荷的改变导致肌纤维表型变化的分子机制。 肌纤维表型和功能的典型标志物是肌球蛋白重链（MyHC），因为它是骨骼肌纤维最大无负荷缩短速度（Vmax）的主要决定因素。 通过建立一个机械负荷降低的小鼠模型[后肢非负重（NWB）]来研究betaMyHC基因的转录调控，我们先前已经在体内鉴定了第一个NWB应答启动子区（-450至-294）。 我们还发现，该NWB区域含有一个负元件（-332至-311;称为dbetaNRE-S），该元件结合仅在NWB比目鱼核提取物中鉴定的两种不同蛋白质。 因此，该建议的主要焦点是鉴定在NWB条件下转录抑制betaMyHC基因表达的核因子，并测试它们在纤维特异性表达（FSE）中的可能作用。 提出的精细水平的实验包括：1）使用表达克隆分离编码结合dbetaNRE-S的核转录因子的cDNA。北方分析将确定核因子的发育、组织和FSE模式。总体水平涉及产生和分析携带以下基因的转基因小鼠：1）携带dbetaNRE-S元件的PCR定点突变的转基因，以确定其作为NWB-E的真实性。 将通过测定NWB-比目鱼肌提取物中的氯霉素乙酰转移酶（CAT）比活性来测量转基因启动子活性，和2）过表达编码特异性靶向横纹肌的核因子的cDNA的转基因。 RNA和蛋白质分析将评估cDNA过表达对整个横纹肌表型的影响，这将与整个肌肉和单纤维功能分析相关。 所提出的实验将鉴定和测试参与NWB诱导的β MyHC基因表达的转录抑制及其可能的FSE的核因子和β MyHC顺式元件的体内功能。 核因子的体内过表达将确定它们在调节其他肌肉基因、肌肉萎缩和发育中的潜在作用。 预计这些实验将确定潜在的DNA/蛋白质靶点，用于针对由疾病、太空飞行或长期卧床休息引起的机械负荷改变引起的慢-快肌肉表型和姿势功能衰弱性丧失提供对策。

One major outstanding question in muscle biology involves determining the molecular mechanisms by which alterations in mechanical load lead to changes in muscle fiber phenotype. A quintessential marker of myofiber phenotype and function is the myosin heavy chain (MyHC) since it is a major determinate of maximum unloaded velocity of shortening (Vmax) of skeletal muscle fibers. By developing a mouse model of decreased mechanical loading [hindlimb nonweight bearing (NWB)] to investigate transcriptional regulation of the betaMyHC gene we have previously identified in vivo the first NWB responsive promoter region (-450 to -294). We also showed that this NWB region contains a negative element (-332 to -311; termed dbetaNRE-S) that binds two different proteins identified ONLY in NWB-soleus nuclear extracts. Thus, the major focus of this proposal is to identify the nuclear factor(s) that transcriptional repress betaMyHC gene expression under NWB conditions, and to test their possible role in fiber-specific expression (FSE). Proposed experiments at the fine level involve: 1) isolating cDNAs encoding nuclear transcription factor(s) that bind the dbetaNRE-S using expression cloning. Northern analysis will determine developmental, tissue and FSE pattern of the nuclear factor(s). The gross level involves the generation and analyses of transgenic mice harboring: 1) a transgene carrying PCR site- directed mutation of the dbetaNRE-S element to determine its authenticity as an NWB-E. Transgene promoter activity will be measured by assaying for chloramphenicol acetyltransferase (CAT) specific activity in NWB-soleus muscle extract, and 2) transgenes overexpressing cDNA(s) encoding nuclear factor(s) specifically targeted to striated muscle. RNA and protein analyses will assess the impact of cDNA overexpression on whole striated muscle phenotype, which will be correlated to whole muscle and single fiber functional analysis. The proposed experiments will identify and test the in vivo function of nuclear factor(s) and betaMyHC cis-element(s) involved in NWB induced transcriptional repression of betaMyHC gene expression and possibly its FSE. In vivo overexpression of nuclear factor(s) will identify their potential roles in regulation of other muscle gene(s), muscle atrophy and development. These experiments are expected to identify potential DNA/protein targets for therapies aimed at providing counter-measures against the slow-to-fast muscle phenotype and debilitating loss of postural function induced by altered mechanical loads resulting from disease, space flight or extended bed rest.