MECHANISM OF CALCIUM OVERLOADING

钙超载的机制

• 批准号: 2219273
• 负责人: TUAN H KUO
• 金额: $ 15.31万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2219273
• 关键词: RNA splicing; autoradiography; calcium binding protein; calcium channel blockers; calcium flux; calcium transporting ATPase; cyclic AMP; cyclic GMP; disease /disorder model; gel electrophoresis; gene expression; genetic disorder; genetic promoter element; hamsters; heart cell; heart disorder; heart pharmacology; immunoprecipitation; insulin; isoproterenol; membrane permeability; molecular cloning; northern blottings; nucleic acid sequence; phosphorylation; polymerase chain reaction; protein kinase A; protein kinase C; radiotracer; sarcolemma; sarcoplasmic reticulum; second messengers; thyroid hormones; tissue /cell culture; transfection

The long term goal of this project is to elucidate the pathogenesis of hereditary cardiomyopathy. Intracellular calcium overload due to changes in the calcium homeostatic mechanisms is a characteristic feature of the hereditary cardiomyopathy in Syrian hamster. It is our hypothesis that a primary defect in the regulation of the plasma membrane Ca2+ pump at the level of gene expression and/or protein phosphorylation can lead to altered Ca2+ hemostasis and overload. In order to test this hypothesis, we propose to characterize the defects in the Ca2+ pump regulation at gene and protein level by comparing the cardiac tissue or myocytes from control and myopathic animals. Specifically, we propose: 1. To study the regulation of Ca2+ pump gene expression. The mRNA level of Ca2+ pump in cultured myocytes will be determined under basal or stimulated conditions. The effect of various agents such as protein kinase C-activator (phorbol ester), protein kinase A activator (isoproterenol, cAMP), as well as Ca2+-ionophore (ionomycin) or hormones (insulin, and thyroid hormone) shall be tested either individually or in combination, using Northern analysis and results correlated with the Ca2+ ATPase activity assay. Furthermore, the promoter of the Ca2+ pump gene will be isolated and characterized by molecular cloning and DNA sequencing. 2. To detect altered RNA splicing by RNA-PCR analysis. Possible changes in the Ca2+ pump mRNA splicing pattern during the development of cardiomyopathy, will be determined by polymerase chain reaction (PCR) analysis. The effect of various stimulators (sp. aim 1) on RNA splicing will also be studied in cultured myocytes. 3. To study the regulation of Ca2+ pump activity by kinase-mediated phosphorylation. The effect of various kinases such as protein kinase C, cAMP-dependent kinase, cGMP-dependent kinase or Ca2+/calmodulin-dependent kinase on Ca2+ pump phosphorylation will be examined in cardiac plasma membranes of the control and myopathic hamsters and results correlated with the Ca2+- ATPase activity assay. Similar analysis will be performed to examine the regulation of the sarcoplasmic reticulum calcium pump via phosphorylation of the phospholamban. Thus this project is not only relevant to the study of pathogenesis of cardiomyopathy but also may contribute to the understanding of mechanisms that regulate the Ca2+ pump protein and its gene expression.

本项目的长期目标是阐明 遗传性心肌病 细胞内钙超载由于变化 在钙稳态机制是一个典型的特点， 叙利亚仓鼠遗传性心肌病 我们假设 一个主要的缺陷，在调节质膜钙泵在 基因表达和/或蛋白质磷酸化水平可导致 改变Ca 2+止血和过载。 为了验证这一假设， 我们建议将Ca 2+泵调节中的缺陷描述为 基因和蛋白质水平，通过比较心脏组织或心肌细胞， 对照和肌病动物。 具体而言，我们建议： 1.研究钙泵基因表达的调控。 将测定培养的心肌细胞中Ca 2+泵的mRNA水平 在基础或刺激条件下。 各种药剂的作用， 作为蛋白激酶C-激活剂（佛波醇酯）、蛋白激酶A激活剂 （异丙肾上腺素，cAMP），以及Ca 2 +-离子载体（离子霉素）或激素 （胰岛素和甲状腺激素）应单独或在 组合，使用北方分析和结果与Ca 2 + ATP酶活性测定。 此外，Ca 2+泵基因的启动子 将通过分子克隆和DNA技术分离和鉴定 测序 2.通过RNA-PCR分析检测RNA剪接的改变。 钙泵mRNA剪接模式的可能变化 心肌病的发展，将通过聚合酶链 反应（PCR）分析。 各种刺激物的作用（sp.aim1） 也将在培养的肌细胞中研究RNA剪接。 3.研究激酶介导的钙泵活性调节 磷酸化 各种激酶如蛋白激酶C、cAMP依赖性 激酶、cGMP依赖性激酶或Ca 2 +/钙调蛋白依赖性激酶对Ca 2 + 泵磷酸化将在心肌细胞膜中进行检查。 对照和肌病仓鼠，结果与Ca 2 +- ATP酶活性测定。 将进行类似的分析， 通过磷酸化调节肌浆网钙泵 受磷兰班 因此，本项目不仅涉及到对 心肌病，但也可能有助于了解机制 调节Ca 2+泵蛋白及其基因表达。