ALTERED MECHANICAL LOADS AND SKELETAL MUSCLE PHENOTYPE

肌肉生物学中一个突出的主要问题是确定机械负荷变化导致肌肉纤维表型变化的分子机制。肌球蛋白重链(MyHC)是肌纤维表型和功能的一个典型标志，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确定了第一个NWB响应启动子区域(-450到-294)。我们还发现，该NWB区含有一个负元件(-332到-311；称为dbetaNRE-S)，它结合了仅在NWB-比目鱼核提取液中发现的两种不同的蛋白质。因此，本研究的重点是确定核因子(S)在NWB条件下转录抑制β-MyHC基因表达，并测试其在纤维特异性表达中的可能作用。拟在精细水平上进行的实验包括：1)利用表达克隆技术分离与核转录因子(S)结合的核转录因子(DNAs)。Northern分析将确定核因子的发育、组织和FSE模式(S)。总体水平包括转基因小鼠的产生和分析：1)携带dbetaNRE-S元件的转基因动物的定点突变，以确定其作为NWB-E的真实性。转基因启动子活性将通过检测比目鱼肌提取物中氯霉素乙酰转移酶(CAT)的特异性活性来检测；2)针对横纹肌的过表达编码核因子的基因(S)(S)。RNA和蛋白质分析将评估cDNA超表达对整个横纹肌表型的影响，这将与整体肌肉和单纤维功能分析相关。拟进行的实验将鉴定和测试核因子(S)和β-MyHC顺式元件(S)在体内参与核转录抑制β-MyHC基因表达及其可能的FSE的功能。核因子在体内的过表达(S)将确定它们在调节其他肌肉基因(S)、肌肉萎缩和发育中的潜在作用。这些实验有望确定潜在的DNA/蛋白质靶点，用于治疗旨在针对由疾病、太空飞行或长时间卧床引起的机械负荷变化引起的肌肉表型由慢变快和衰弱性丧失的反措施。

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.