BETA-ADRENERGIC REGULATION OF A MYOCARDIAL ACTIN GENE

心肌肌动蛋白基因的β-肾上腺素能调节

基本信息

批准号：
2423722
负责人：
Nanette Hahr Bishopric
金额：
$ 21.7万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-01-01 至 1998-12-31
项目状态：
已结题

项目摘要

Beta-adrenergic agonists are strongly implicated in myocardial hypertrophy in vivo, and beta-adrenergic and cyclic AMP (cAMP)-dependent mechanisms are a frequent target of drugs used in ischemic cardiovascular disease and heart failure. However, the mechanisms transducing beta-adrenoceptor- mediated myocardial cell growth and gene expression are poorly understood. With few exceptions, beta-adrenergic effects in the heart have been thought to be mediated via modulation of cAMP. We have recently identified a novel pathway for beta-adrenergic gene regulation in cardiac myocytes that appears to be coupled to the storage or flux of calcium from physically or biochemically defined compartments and is independent of cAMP and cAMP-dependent protein kinase. The skeletal alpha-actin (sACT) gene, encoding a developmentally regulated actin-actin isoform, is selectively regulated by this pathway during beta-adrenoceptor-mediated hypertrophy. In order to identify essential components of this signal transduction mechanism, we propose to identify pharmacologic mechanisms for beta-adrenergic regulation of the sACT gene and to examine interactions between the sACT promoter and DNA-binding transcriptional activating factors that may be required for beta-adrenergic induction. Experiments described in part (1) will evaluate the role of beta- adrenergic, protein kinase A-independent, and calcium-dependent mechanisms in transcriptional and posttranscriptional regulation of the sACT gene, employing nuclear run-on and messenger RNA half-life assays. Because we have determined that transcriptional activating factors Fos and Jun (AP-1) positively regulates sACT in cardiac myocytes, we will determine the effects of protein kinase A-dependent, and calcium-dependent mechanisms on expression of c-fos, c-jun, and related genes as well as on AP-1 binding activity and immunoreactivity. Pl9 teratocarcinoma cells will be stably transfected with an sACT/lacZ chimeric gene containing full-length promoter sequences, most coding sequences and 3' flanking sequences in order to create a cell line in which the signal-mediated regulation of this gene can be studied directly. In part (2), beta-adrenergic and AP- 1- responsive bases in the proximal sACT promoter will be determined by a combination of in situ mutagenesis, gel mobility retardation assay, and methylation interference. Cardiac myocyte nuclear proteins will be evaluated for interaction with the sACT promoter, and a potential interaction between serum response factor and AP-1 on this promoter will be assessed. Finally, lambda-gt11 cardiac myocyte cDNA expression libraries will be constructed and screened with relevant oligonucleotide sequences from the sACT promoter.

β-肾上腺素能激动剂与心肌肥大有关体内，β-肾上腺素能和环状AMP（CAMP）依赖性机制是缺血性心血管疾病和心脏衰竭。但是，转导β-肾上腺素受体的机制 - 介导的心肌细胞生长和基因表达知之甚少。除少数例外，心脏中的β-肾上腺素能作用是被认为是通过训练营的调节而介导的。我们最近确定了心肌细胞中β-肾上腺素能基因调节的新途径似乎与从物理或生化定义的隔室，独立于营地和cAMP依赖性蛋白激酶。骨骼α-肌动蛋白（SACT）基因编码开发调节的肌动蛋白 - 肌动蛋白，IS 在β-肾上腺肾上腺介导的期间由该途径有选择性地调节肥大。为了识别此信号的基本组件转导机制，我们建议识别药理机制用于β-肾上腺素能调节，并检查 SACT启动子与DNA结合转录之间的相互作用 β-肾上腺素能诱导可能需要的激活因子。第（1）部分中描述的实验将评估β- 肾上腺素，蛋白激酶A非依赖性和钙依赖性机制在SACT基因的转录和转录后调节中采用核跑步和Messenger RNA半衰期测定法。因为我们已经确定转录激活因子FOS和JUN（AP-1）积极调节心肌细胞中的sact，我们将确定蛋白激酶A依赖性和钙依赖性机制对 C-FOS，C-JUN和相关基因以及AP-1结合的表达活性和免疫反应性。 PL9畸胎瘤细胞将稳定用含有全长的sact/lacz嵌合基因转染启动子序列，大多数编码序列和3'侧翼序列为了创建一个单元线，信号介导的调节该基因可以直接研究。在第（2）部分中，β-肾上腺素能和AP-1- 近端SACT启动子中的响应基础将由A确定原位诱变，凝胶迁移迟缓测定和甲基化干扰。心肌细胞核蛋白将是评估与SATS启动子的相互作用，并且潜力该启动子上血清反应因子与AP-1之间的相互作用将被评估。最后，lambda-gt11心肌细胞cDNA表达图书馆将通过相关的寡核苷酸构建和筛选 SACT启动子的序列。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Modulation of cytokine-induced cardiac myocyte apoptosis by nitric oxide, Bak, and Bcl-x.

DOI：
10.1161/01.res.84.1.21
发表时间：
1999-01
期刊：
Circulation research
影响因子：
20.1
作者：
Douglas J. Ing;Jie Zang;Victor J. Dzau;Keith A. Webster;N. Bishopric
通讯作者：
Douglas J. Ing;Jie Zang;Victor J. Dzau;Keith A. Webster;N. Bishopric

Fusigenic liposome-mediated DNA transfer into cardiac myocytes.

融合脂质体介导的 DNA 转移到心肌细胞中。

DOI：
10.1006/jmcc.1996.0130
发表时间：
1996
期刊：
Journal of molecular and cellular cardiology.
影响因子：
0
作者：
Ellison,KE;Bishopric,NH;Webster,KA;Morishita,R;Gibbons,GH;Kaneda,Y;Sato,B;Dzau,VJ
通讯作者：
Dzau,VJ

Regulated expression of a foreign gene targeted to the ischaemic myocardium.

针对缺血心肌的外源基因的调节表达。