BETA-ADRENERGIC REGULATION OF A MYOCARDIAL ACTIN GENE

β-肾上腺素能激动剂与心肌肥厚密切相关 体内β-肾上腺素能和环磷酸腺苷(CAMP)依赖机制 是治疗缺血性心血管疾病的药物的常见靶点， 心力衰竭。然而，β-肾上腺素能受体的转导机制- 介导的心肌细胞生长和基因表达目前还知之甚少。 除了极少数例外，心脏中的β-肾上腺素能效应 被认为是通过调节cAMP来调节的。我们最近确认了 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，独立于 CAMP和cAMP依赖的蛋白激酶。骨骼α肌动蛋白(SACT) 该基因编码一种发育调节的肌动蛋白-肌动蛋白亚型，是 在β-肾上腺素能受体介导的过程中受该途径的选择性调节 肥大。为了识别该信号的基本成分 转导机制，我们建议识别药理机制 对sACT基因进行β-肾上腺素能调节，并检测 SACT启动子与DNA结合转录的相互作用 激活β-肾上腺素能诱导所需的因子。 第(1)部分中描述的实验将评估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控中， 使用核连续和信使RNA半衰期分析。因为我们 已确定转录激活因子Fos和Jun(AP-1) 正向调节心肌细胞中sACT，我们将确定 蛋白激酶A依赖和钙依赖机制对血管内皮细胞生长的影响 C-fos、c-jun及其相关基因在AP-1结合中的表达 活性和免疫反应性。PL9畸胎癌细胞将稳定 含全长sACT/LacZ嵌合基因的转基因 启动子序列、大部分编码序列和3‘侧翼序列 以建立一种细胞系，在该细胞系中，信号介导的调节 这个基因可以直接研究。在第(2)部分，β-肾上腺素能和AP-1- 近端sACT启动子中的反应碱基将由 原位诱变、凝胶迁移率抑制试验和 甲基化干扰。心肌细胞核蛋白将是 评估与sACT启动子的相互作用，以及潜在的 血清反应因子与AP-1在该启动子上的相互作用 被评估。最后，lambda-gt11在心肌细胞中的表达 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.