MECHANISM OF CELL DEATH IN CARDIOMYOPATHY

心肌病细胞死亡的机制

• 批准号: 2028350
• 负责人: TUAN H KUO
• 金额: $ 17.65万
• 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 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2028350
• 关键词: apoptosis; autoradiography; calcium binding protein; calcium channel blockers; calcium flux; calcium transporting ATPase; cell cycle; cyclic AMP; gene expression; genetic disorder; hamsters; heart cell; heart pharmacology; immunoprecipitation; in situ hybridization; laboratory rat; molecular cloning; molecular pathology; myocardium disorder; northern blottings; pathologic process; phosphorylation; polymerase chain reaction; sarcoplasmic reticulum; tissue /cell culture; transfection

DESCRIPTION (Adapted from Investigator's Abstract): The long term goal of this project is to elucidate the pathogenesis of hereditary cardiomyopathy in human. Intracellular Ca 2+ overload and spontaneous myocytolysis are characteristics of the cardiomyopathy in Syrian hamsters. In this animal model, they have shown previously that a simultaneous reduction of the expression of plasma membrane Ca 2+ pump (PMCA) and sarcoplasmic reticulum Ca 2+ pump (SERCA) may be involved in myocyte death. They have also obtained preliminary evidence that apoptosis occurs in myopathic heart, which is accompanied by altered expression of the genes involved in cell cycle (and apoptosis). Based on these evidence, they hypothesize that this cardiomyopathy involves altered Ca 2+ homeostasis which leads to aberrant cell cycle gene expression, cell cycle activation, and apoptosis. They propose: 1. To study the coordinated regulation of PMCA and SERCA genes. PMCA and SERCA genes will be separately transferred into rat adult cardiomyocyte in culture by using a novel adenovirus system. The effect of the exogenous gene transfer on [Ca 2+ ]i homeostasis and on the expression of endogenous PMCA, SERCA, and Ca 2+ channels will be examined. Furthermore, the effect of [Ca 2+ ]i on PMCA and SERCA gene expression will be studied in normal, uninfected myocyte. 2. To study the role of Ca2+ in apoptosis and to test the protective effect of Ca2+ pump. Apoptosis will be induced in cultured myocytes using SERCA inhibitor thapsigargin (Tg) and the expression of cell cycle regulatory genes (c-myc, cyclin A, p53, p21 and bcl-2) will be assayed. The protection of myocyte from Tg-induced apoptosis will be tested by overexpression of PMCA via gene transfer. 3. To study the molecular mechanism of apoptosis in cardiomyopathy. They will examine the development of apoptosis in myopathic heart using in situ terminal deoxynucleotidyl transferase staining. They will determine altered mRNA and protein expression of the cell cycle regulators in isolated heart as well as myocyte. The results will be verified in myopathic heart by in situ hybridization and immunostaining of these markers. DNA synthesis activity will be determined by BrdU labeling of myocytes during disease development. Intervention of the cardiomyopathy will be tested by treatment with calcium antagonist, and heart tissue sections examined for the prevention of lesion development and for the restoration of normal expression pattern of the cell cycle regulators. Thus this project is not only relevant to the study of pathogenesis of cardiomyopathy but also may contribute to the understanding of mechanisms that control apoptosis.

描述（改编自研究者摘要）： 本课题旨在阐明遗传性心肌病的发病机制 在人类中。 细胞内钙超载和自发性肌细胞溶解是 叙利亚仓鼠心肌病的特征。 在该动物中 模型，他们以前已经表明，同时减少 质膜钙泵和肌浆网的表达 钙泵（SERCA）可能参与心肌细胞死亡. 他们还 获得了细胞凋亡发生在肌病心脏的初步证据， 这伴随着细胞内基因表达的改变， 细胞周期（和凋亡）。 根据这些证据，他们假设， 心肌病涉及改变的Ca 2+稳态， 细胞周期基因表达、细胞周期激活和细胞凋亡。 他们 建议：1. 研究PMCA和SERCA基因的协同调控。 将PMCA和SERCA基因分别转入成年大鼠 心肌细胞的培养，使用新的腺病毒系统。 的影响 外源基因转移对细胞内[Ca ~（2+）]i稳态和细胞内Ca ~（2+）表达的影响 检测内源性PMCA、SERCA和Ca 2+通道。 此外，[Ca 2+ ]i对PMCA和SERCA基因表达的影响也可能与[Ca 2+ ]i对PMCA和SERCA基因表达的影响有关。 在正常未感染的心肌细胞中进行研究。 2. 研究Ca ~（2+）在 检测钙泵的保护作用。 凋亡将是 使用SERCA抑制剂毒胡萝卜素（Tg）在培养的肌细胞中诱导， 细胞周期调控基因（c-myc、cyclin A、p53、p21和 bcl-2）进行测定。 TG诱导的心肌细胞凋亡的保护作用 将通过基因转移过表达PMCA进行检测。 3. 研究 心肌病细胞凋亡的分子机制。 他们将检查 原位末端标记法检测心肌细胞凋亡的研究 脱氧核苷酸转移酶染色。 他们将确定改变的mRNA， 在离体心脏中细胞周期调节因子的蛋白表达以及 肌细胞 本研究结果将在心肌病心脏中通过原位心肌细胞移植进行验证 这些标记的杂交和免疫染色。 DNA合成活性 将通过疾病发展过程中肌细胞的BrdU标记来确定。 将通过钙治疗来测试心肌病的干预 拮抗剂和心脏组织切片检查，以预防病变 发育和恢复细胞的正常表达模式 循环调节器。 因此，该项目不仅与研究有关， 心肌病的发病机制，但也可能有助于了解 控制细胞凋亡的机制。