SUBCELLULAR MRNA DISTRIBUTION IN MYOCYTES

适应对于超负荷或衰竭的心脏来说是至关重要的，因为 心脏中的总肌原纤维质量决定了心脏的力量。我的 长期目标是了解横纹肌如何适应其分子 对生理需求的变化作出反应的成分。至 剖析刺激和肥厚之间的机制，我很早就研究了 在第一个小时内发生的变化。我的假设是，早些时候 生长受两个过程控制；主要的 转录因子与细胞质mRNA的调控。这项研究需要 对β-肾上腺素能途径的快速药物干预。 因此，我使用了新生大鼠心脏的原代细胞培养。这 不同的、混合的细胞群体最好用细胞生物学来研究 单细胞水平的技术。我用免疫化学来监测 转录因子磷酸化激活，cAMP调节 元素结合蛋白(CREB)。我用原位杂交技术来检测 亚蜂窝消息分发的变化。特定目标1的特点是 CREB的磷酸化途径，决定了一种有效的 PKA激活促进细胞肥大，并创造条件 增加肌原纤维中的α-肌球蛋白沉积。具体目标2 量化核内信使核糖核酸的向外迁移并区分 信使核糖核酸的分布模式是由于扩散、运输还是 翻译陷阱。特定目标3测试非翻译区， 3‘非编码区，是微管运输或翻译控制所必需的。 我使用了β-半乳糖苷酶编码的嵌合结构的转染法 区域和α-肌球蛋白3‘非编码区。细胞质mRNA的研究尚未见报道 充分探讨作为心肌肥厚的一种快速调节事件。

Adaptation is of utmost importance in overloaded or failing hearts because the total myofibrillar mass in the heart determines cardiac strength. My long-term goal is to understand how striated muscle adapts its molecular composition in response to changes in its physiological demands. To dissect mechanisms between stimulation and hypertrophy, I examine early changes occurring in the first hour. My hypotheses proposes that early growth is governed by two processes; phosphorylation of a major transcription factor and control of cytoplasmic mRNA. The study requires rapid pharmacological interventions to the beta-adrenergic pathway. Therefore, I use a primary cell culture from neonatal rat heart. This diverse, mixed cell population is best studied by cell biological techniques at the single cell level. I use immunochemistry to monitor activation by phosphorylation of the transcription factor, cAMP regulatory element binding protein (CREB). I use in situ hybridization to examine changes in subcellular message distribution. Specific aim 1 characterizes the pathways of CREB phosphorylation, determines an effective paradigm of PKA activation for cell hypertrophy, and establishes conditions that increase alpha-myosin protein deposition in myofibrils. Specific aim 2 quantifies the outward migration of mRNA from the nucleus and distinguishes whether the mRNA distribution pattern is due to diffusion, transport, or translational trapping. Specific aim 3 tests if the untranslated region, 3'UTR, is required for transport by microtubules or control of translation. I use transfection with a chimeric construct of beta-galactosidase coding region and alpha-myosin 3'UTR. Study of cytoplasmic mRNA has not been sufficiently explored as a rapid regulatory event in cardiac hypertrophy.