EXERCISE HYPERTROPHY AND CONTROL OF MYOSIN INDUCTION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 机械超负荷（MOV）导致成年横纹肌肥大，肌球蛋白表型和收缩特性发生明显变化。成人阶段肌肉生物学中剩余的关键问题涉及确定纤维类型特异性表达（FSE）和MOV诱导的肌肉基因表达变化所需的DNA调节元件和核蛋白因子。这个提议？本研究的主要目的是鉴定转录诱导MOV跖肌β-肌球蛋白重链（bMHC）表达的DNA元件和核因子，并检测它们在FSE中的可能作用。我们提出了两个层次的调查实验。罚款水平包括：1）使用迁移率变动、DNA酶I足迹法和甲基化干扰测定，鉴定核因子和DNA元件之间的相互作用，所述相互作用显示为MOV体内诱导bMHC所必需的，和2）使用表达克隆分离编码参与MOV诱导bMHC的核因子的cDNA。北方分析将确定这些核因子的发育、组织和FSE模式。总体水平涉及转基因小鼠的产生和分析，所述转基因小鼠具有：1）携带参与MOV诱导bMHC的DNA元件的PCR定点突变的转基因。通过测定MOV后和含有各种纤维类型的肌肉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。北方分析将评估核因子过表达对横纹肌表型的影响。拟开展的工作将鉴定和测试参与bMHC FS和MOV诱导的核因子和bMHC启动子元件的体内功能。核因子的体内过表达将确定它们在调节其他基因、肌肉增大和发育中的潜在作用。预计这些实验将确定用于治疗的潜在蛋白质/DNA靶点，旨在为肌肉疾病引起的肌肉表型和生长模式紊乱提供对策。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mechanical overload (MOV) leads to enlargement of adult striated muscle and distinct changes in both myosin phenotype and contractile properties. Remaining critical questions in adult-stage muscle biology involve determining the DNA regulatory element(s) and nuclear protein factor(s) required for fiber-type specific expression (FSE) and MOV induced changes in muscle gene expression. This proposal?s major focus is to identify the DNA element(s) and nuclear factor(s) which transcriptionally induce beta myosin heavy chain (bMHC) expression in MOV plantaris muscle and to test their possible role in FSE. We propose experiments for two levels of inquiry. The fine level involves: 1) identifying interactions between nuclear factor(s) and DNA element(s) shown to be necessary for in vivo induction of bMHC by MOV, using mobility shift, DNAse I footprinting and methylation interference assays, and 2) isolating cDNA(s) encoding nuclear factor(s) involved in bMHC induction by MOV using expression cloning. Northern analysis will determine developmental, tissue and FSE patterns of these nuclear factor(s). The gross level involves the genera-tion and analysis of transgenic mice harboring: 1) transgenes carrying PCR site-directed mutations of DNA element(s) involved in bMHC induction by MOV. Transgene promoter activity will be measured by assaying for chloramphenicol acetyltransferase (CAT) specific activity following MOV and in muscles containing various fiber types, and 2) transgenes over-expressing cDNA(s) encoding nuclear factor(s) specifically targeted to striated muscle. Northern analysis will assess the impact of overexpression of nuclear factors on striated muscle phenotype. The proposed work will identify and test the in viv function of nuclear factor(s) and bMHC promoter element(s) involved in bMHC FS and induction by MOV. In vivo overexpression of nuclear factor(s) will identif their potential roles in regulation of other gene(s), muscle enlargement and development. These experiments are expected to identify potential protein/DNA targets for therapies aimed at providing countermeasures to derangements in muscle phenotypes and growth patterns attributable to muscle diseases.