EXERCISE HYPERTROPHY AND CONTROL OF MYOSIN INDUCTION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 机械过载(MOV)导致成人横纹肌增大，肌球蛋白表型和收缩特性均发生明显变化。成体阶段肌肉生物学的关键问题包括确定纤维类型特异性表达和MOV诱导的肌肉基因表达变化所需的DNA调控元件(S)和核蛋白因子(S)。S的主要工作是确定转录上诱导MOV足底肌β肌球蛋白重链(BMHC)表达的DNA元件(S)和核因子(S)，并检测它们在肌肉萎缩性侧索硬化症中的可能作用。我们提出了两个层次的调查实验。精细水平包括：1)利用迁移率改变、DNase I足迹和甲基化干扰分析，鉴定在MOV体内诱导bMHC所必需的核因子(S)与DNA元件(S)之间的相互作用；2)利用表达克隆技术分离编码参与MOV诱导bMHC的核因子(S)的基因。Northern分析将确定这些核因子的发育、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MOV诱导bMHC基因定点突变的转基因小鼠(S)。转基因启动子活性将通过检测MOV后和含有不同类型纤维的肌肉中氯霉素乙酰转移酶(CAT)的特异性活性来测量；2)针对横纹肌的过表达编码核因子的基因(S)(S)。Northern分析将评估核因子过度表达对横纹肌表型的影响。这项工作将鉴定和测试参与bMHC FS和MOV诱导的核因子(S)和bMHC启动子元件(S)的在体功能。体内核因子的过表达(S)将确定它们在调节其他基因(S)、肌肉增大和发育中的潜在作用。这些实验有望确定潜在的蛋白质/DNA靶点用于治疗，旨在为肌肉疾病引起的肌肉表型和生长模式的紊乱提供对策。

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.