BETA-ADRENERGIC REGULATION OF A MYOCARDIAL ACTIN GENE

β-肾上腺素能受体激动剂与心肌肥厚密切相关 在体内，β-肾上腺素能和环腺苷酸（cAMP）依赖性机制 是缺血性心血管疾病药物的常见靶点， 心衰然而，β肾上腺素受体的转导机制- 介导的心肌细胞生长和基因表达知之甚少。 除了少数例外，心脏中的β-肾上腺素能作用一直是 被认为是通过调节cAMP介导的。我们最近发现 心肌细胞β-肾上腺素能基因调控的新途径 这似乎与钙的储存或流动有关， 物理或生物化学定义的隔室，并且独立于 cAMP和cAMP依赖性蛋白激酶。骨骼α-肌动蛋白（sACT） 基因，编码发育调节肌动蛋白-肌动蛋白亚型， 在β-肾上腺素受体介导的 肥厚为了识别这个信号的基本成分 转导机制，我们建议确定药理学机制 β-肾上腺素能调节sACT基因， sACT启动子和DNA结合转录因子之间的相互作用 β-肾上腺素能诱导可能需要的激活因子。 第（1）部分描述的实验将评估β- 肾上腺素能、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， 使用核连续和信使RNA半衰期测定。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和相关基因的表达以及AP-1结合 活性和免疫反应性。P19畸胎瘤细胞将稳定地生长。 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， 为了建立一个细胞系，其中信号介导的调节 这个基因可以直接研究。在第（2）部分中，β-肾上腺素能和AP-1- 在近端sACT启动子中的响应碱基将通过免疫组织化学方法确定。 原位诱变、凝胶迁移率阻滞测定和 甲基化干扰心肌细胞核蛋白将被 评估与sACT启动子的相互作用， 血清应答因子和AP-1在该启动子上相互作用将 进行评估。最后，检测了心肌细胞cDNA表达 将构建文库并用相关寡核苷酸进行筛选 来自sACT启动子的序列。

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.