OXIDATIVE PHOSPHORYLATION IN CARDIAC HYPERTROPHY

心脏肥大中的氧化磷酸化

• 批准号: 2668754
• 负责人: TERRENCE Xavier O'BRIEN
• 金额: $ 9.52万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2668754
• 关键词: adenosinetriphosphatase; cats; cytochrome c; cytochrome oxidase; disease /disorder model; enzyme activity; genetic enhancer element; genetic promoter element; genetic transcription; laboratory rat; messenger RNA; mitochondria; northern blottings; oxidative phosphorylation; polymerase chain reaction; posttranscriptional RNA processing; receptor expression; tissue /cell culture; transfection; ventricular hypertrophy; western blottings

To eventually understand the mechanisms of cardiac hypertrophic stimulation and its subsequent progression to congestive heart failure, sense must be made of a complex cascade of hemodynamic and molecular events that affect the cardiocyte's ability to function. The preliminary data generated by the applicant establishes that among the cardiocytes initial response to pathologic hemodynamic load, both in vivo and in vitro, is an up regulation of several components of the nuclear and mitochondrially derived mitochondrial oxidative phosphorylation system at both the mRNA and protein level. Initial studies in a feline pressure- overloaded right ventricle model show upregulation within l to 4 hours of the FO6, beta-F1, and alpha-F1, subunits of FOF1-ATP synthase, as well as cytochrome b and cytochrome oxidase subunits Il and IV. These novel findings have been confirmed in two in vitro neonatal rat cardiocyte models of hypertrophic stimulation, one using electrical pacing and one with phenylephrine. These models have also demonstrated an increase in cytochrome c at the protein level. Oncemore, cloning 1480 nucleotides of the 5' flanking region of the critical beta-F1 ATPase gene followed by transient transfection of a reporter construct into neonatal cardiocytes demonstrates a cis region which upregulates in response to phenylephrine and electrical pacing. As the link of early transcriptional (and/or post-) events in the cardiocyte which may lead to physiologically relevant responses to acute load are largely unknown, this proposal offers a strategy for an understanding of the coordinated expression of the nuclear and mitochondrial genomes in this unique phenotype along 4 specific aims: Specific Aim l: To define the response of highly selected nuclear and mitochondrially encoded oxidative phosphorylation gene mRNAs and their proteins involved in the cardiocyte's response to acute and chronic hypertrophic stress. In particular, the mitochondrially encoded FO6 and nuclear encoded beta-Fl ATPase and cytochrome c will be studied. Partial cDNA constructs and characterized antibodies (to the nuclear gene products) have been used to generate the preliminary data. Specific Aim 2: To determine the relative importance of transcriptional versus post-transcriptional mechanisms in the acute upregulation of mitochondrial mRNAs involved in the cardiocyte's hypertrophic response. The beta-Fl ATPase subunit contains a critical catalytic site and is an excellent candidate to study response in the cardiocyte. Preliminary data indicates much of the response is transcriptional. Specific Aim 3: To determine the promoter-enhancer regions of the beta- ATPase gene important in the initiation and regulation of the cardiocyte's hypertrophic response. Specific Aim 4: To confirm in well-characterized adult models of pressure overload the regulatory response elements determined to be involved in the cardiac hypertrophic response in vitro. Once elements have been identified using the in vitro models, the in vivo response to hypertrophic load stimulation will be determined by established methods of direct gene transfer into the feline heart before and after pulmonary artery banding. As confirmation in cultured, primary adult cardiocytes, highly selected constructs would be transfected using replication-deficient adenovirus constructs. Studying the genetic regulation of oxidative phosphorylation in cardiocytes subjected to hypertrophic stimulus should lead to important, physiologically relevant, insights into key initial molecular events.

最终了解心脏肥厚的机制 刺激及其随后发展到充血性心力衰竭， 感觉必须由复杂的血流动力学和分子级联 影响心电细胞功能能力的事件。 初步 申请人生成的数据确定了心脏细胞中的数据 对病理血流动力学负荷的初步反应，无论是体内还是在体内 体外，是对核的几个组成部分的加强调节 线粒体衍生的线粒体氧化磷酸化系统在 mRNA和蛋白质水平。猫压力的初步研究 右心室模型超负荷显示在L以内的L至4小时以内 FOF1-ATP合酶的FO6，Beta-F1和Alpha-F1，以及 细胞色素B和细胞色素氧化酶亚基IL和IV。这些小说 在两个体外新生儿大鼠心脏细胞中已经确认了发现 肥厚刺激的模型，一种使用电气起诉和一个 与苯肾上腺素。这些模型也显示出增加 蛋白质水平的细胞色素c。 Oncemore，克隆1480个核苷酸的克隆 关键β-F1 ATPase基因的5'侧面区域，然后是 将记者构造转染到新生儿心脏细胞 展示一个顺式区域，该区域会响应于苯肾上腺素 和电气起诉。作为早期转录（和/或后）的链接 心脏细胞中可能导致生理相关的事件 对急性负荷的反应在很大程度上未知，该建议提供了 了解核的协调表达的策略 沿着4个特定目的，这种独特表型中的线粒体基因组： 特定目的l：定义高度选择的核和 线粒体编码的氧化磷酸化基因mRNA及其它们的 蛋白质参与心脏细胞对急性和慢性的反应 肥厚应激。特别是，线粒体编码的FO6和 将研究核编码的β-FL ATPase和细胞色素C。部分的 cDNA构建体和表征抗体（核基因 产品）已用于生成初步数据。 特定目的2：确定转录的相对重要性 在急性上调的转录机制与后期机制 线粒体mRNA参与心脏细胞的肥厚性反应。 β-FL ATPase亚基包含一个关键的催化位点，是一个 在心脏细胞中研究反应的出色候选人。初步数据 指示大部分响应是转录的。 特定目的3：确定β- ATPase基因对心细胞的启动和调节很重要 肥厚的反应。 特定目的4：在特征良好的成人压力模型中确认 超载确定与 体外心脏肥厚反应。一旦确定了元素 使用体外模型，体内对肥厚载荷的反应 刺激将通过已建立的直接基因方法确定 肺动脉束带前后转移到猫心。 作为培养的原代成人心细胞的确认，高度选择 构建体将使用缺乏复制的腺病毒转染 构造。 研究氧化磷酸化的遗传调节 受肥厚刺激的心细胞应导致重要， 生理上相关的，对关键初始分子事件的见解。