REGULATION OF MYOSIN HEAVY CHAIN ALPHA GENE BY CAMP

所提出的实验是我们对 在正常发育过程中和改变的心脏表型的决定因素 心脏的功能状态。 由于cAMP在 心肌收缩力，我们将研究其在α-肌球蛋白的调制作用 重链（MHC）表达。 我们的初步研究表明， 毛喉素诱导稳定状态水平的数倍增加， 血清预处理胎鼠原代培养物中MHC-α mRNA的表达 而在MHC-beta的丰度中没有观察到变化 成绩单。 因此，我们将主要集中在转录 α-MHC基因的调控。 我们将首先完成 通过比较培养的心肌细胞中cAMP反应的特征， mRNA的稳定水平及其转录速率，并通过评估mRNA 稳定 其次，为了阐明血清诱导cAMP的机制， 转录调控，我们将研究启动子活性使用 原代肌细胞培养物的瞬时转染测定。两者 调控序列及其同源转录因子 将研究血清诱导的cAMP效应，并研究其可能的 互动评估。 因此，我们的具体目标是：1。探讨 cAMP对MHC-α基因表达的调节水平。a.分析 cAMP对MHC-α基因表达的转录调控。B.确定 cAMP对 MHC-alpha mRNA稳定性。2.研究启动子响应 营a.描绘cAMP反应元件（AP-2 E）和血清反应 MHC-alpha启动子内的SRE元件。B.研究各自的作用 转录因子AP-2和SRF对MHC-alpha基因表达的影响。

The proposed experiments are a continuation of our studies on the determinants of cardiac phenotype during normal development and in altered functional states of the heart. Since cAMP plays an important role in cardiac contractility, we will study its role in modulation of alpha-myosin heavy chain (MHC) expression. Our preliminary studies have shown that forskolin induces a several fold increase in the steady state level of MHC-alpha mRNA in serum-pretreated primary cultures derived from fetal rat hearts while no change was observed in the abundance of MHC-beta transcript. Therefore, we will focus primarily on the transcriptional regulation of the alpha-MHC gene. We will first complete our ongoing characterization of the cAMP response in cultured myocytes by comparing the steady level of mRNA with its transcription rate and by evaluating the mRNA stability. Next, to delineate the mechanism(s) of serum induced cAMP transcriptional regulation, we will study the promoter activity using transient transfection assays of primary myocyte cultures. Both the regulatory sequences and their cognate transcription factor(s) responsible for the serum induced cAMP effect will be investigated and their possible interaction evaluated. Thus, our specific aims are to: 1. Investigate the level of regulation of MHC-alpha gene expression by cAMP. a. Analyze the cAMP transcriptional regulation of MHC-alpha gene expression. b. Determine the effect of cAMP on MHC-alpha mRNA stability. 2. Study the promoter function in response to cAMP. a. Delineate the cAMP responsive element (AP-2E) and serum response element (SRE) within MHC-alpha promoter. b. Study the role of respective transcription factors, AP-2 and SRF on the MHC-alpha gene expression.