Mitochondria/Mass Spectroscopy Core

线粒体/质谱核心

• 批准号: 7750211
• 负责人: Charles Leslie Hoppel
• 金额: $ 28.67万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-14 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7750211
• 关键词: Affect; Attention; Canis familiaris; Cardiac; Cardiolipins; Cells; Complex; Electron Transport; Electrophoresis; Energy Metabolism; Functional disorder; Heart; Heart failure; Individual; Instruction; Isoprostanes; Lipids; Malonyl Coenzyme A; Mass Spectrum Analysis; Measures; Membrane; Mitochondria; Modification; Mus; Muscle Cells; Myocardium; Myofibrils; Oxidative Stress; Phospholipids; Physiological; Population; Program Research Project Grants; Quality Control; Sampling; Services; Skin; Tissues; Transmission Electron Microscopy; Trypsin; acylcarnitine; analytical method; complex IV; in vivo; oxidation; oxidized low density lipoprotein; research study

Cardiac mitochondria exist in two distinct populations wathin the myocyte, subsarcolemmal mitochondria (SSM), located beneath the sarcolemmal membrane, and interfibrillar mitochondria (IFM) that reside between the myofibrils. SSM are released by tissue homogenization, whereas the liberation of IFM requires a brief exposure of the resulting skinned myocytes to trypsin treatment. The two populations of mitochondria are differently affected under both physiological and pathophysiological conditions and thus requires to study of each individual population separately. The Mitochondrial/Mass Spec Core (Core D) will perform the following experiments as a service to the different Projects. 1. Isolate subsarcolemmal and interfibrillar mitochondria from experimental hearts; assess quality control. 2. Obtain samples of myocardium and isolated populations of SSM and IFM for electron transport chain complex analysis. 3. Obtain samples of heart, SSM, and IFM for lipid extraction and phospholipid analysis. Special attention is paid to cardiolipin (CL) analysis: CL content, CL species and CL oxidation. 4. Aquire heart tissue for analysis of acyl-CoAs and acylcarnitines with special emphasis on malonyl-CoA a key metabolite in switch in fuel utilization. 5. Carry out mass spectrometric analysis to identify posttranslational and oxidative modifications on ETC subunits, especially complex IV. 6. Perform structural analysis on cardiac tissue and cardiac SSM and IFM by transmission electron microscopy. 7. Isoprostane and oxidized low density lipoprotein (oxLDL) quantitation as a measure of in vivo oxidative stress. 8. Blue Native Electrophoresis will be done on mitochondria from Project 1 and 4. RELEVANCE (See instructions): Mitochondrial are the powerhouses and the cell. During heart failure an energy crises occurs in the heart and is related to the dysfunction of mitochondria. This Core will provide mitochondrial isolation from heart of dogs and mice under experimental conditions and perform analytical methods to support the 4 project within the Program Project Grant.

心肌细胞中的线粒体存在于两个不同的群体中：肌膜下线粒体（SSM）、 位于肌膜下方，纤维间线粒体（IFM）位于肌膜之间 肌原纤维。 SSM 通过组织均质化释放，而 IFM 的释放需要短暂暴露 将所得的去皮肌细胞进行胰蛋白酶处理。两个线粒体群体不同 在生理和病理生理条件下都会受到影响，因此需要研究每一种 单独的人口。线粒体/质谱核心 (Core D) 将执行以下操作 实验作为不同项目的服务。 1. 从实验心脏中分离肌膜下和纤维间线粒体；评估质量控制。 2. 获取心肌样本以及电子传递链复合体的 SSM 和 IFM 分离群体 分析。 3. 获取心脏、SSM 和 IFM 样本，用于脂质提取和磷脂分析。特别注意 心磷脂 (CL) 分析：CL 含量、CL 种类和 CL 氧化。 4. 获取心脏组织用于分析酰基辅酶A和酰基肉碱，特别强调丙二酰辅酶A是关键 燃料利用率转换中的代谢物。 5. 进行质谱分析以鉴定 ETC 上的翻译后和氧化修饰 亚基，尤其是复合体 IV。 6. 通过透射电子显微镜对心脏组织和心脏 SSM 和 IFM 进行结构分析。 7. 异前列烷和氧化低密度脂蛋白 (oxLDL) 定量作为体内氧化的测量 压力。 8. 将对项目 1 和 4 的线粒体进行蓝色天然电泳。 相关性（参见说明）： 线粒体是动力室和细胞。心力衰竭期间，心脏会出现能量危机 且与线粒体功能障碍有关。该核心将提供线粒体与心脏的分离 狗和小鼠在实验条件下进行分析方法以支持4个项目 计划项目补助金。