Mitochondria/Mass Spectroscopy Core

心肌线粒体存在于肌细胞内的两个不同的群体中，即肌膜下线粒体(SSM)， 位于肌膜下的纤维间线粒体(IFM)位于 肌原纤维。SSM是通过组织匀浆释放的，而IFM的释放需要短暂的曝光 用胰酶处理所得的去皮心肌细胞。这两种线粒体是不同的 在生理和病理生理条件下都受到影响，因此需要研究每一种 单独的个体种群。线粒体/质量谱核心(核心D)将执行以下操作 作为服务于不同项目的实验。 1.从实验心脏分离肌膜下和纤维间线粒体，进行质量控制。 2.获取心肌标本和分离的SSM和IFM电子传递链复合体 分析。 3.获取心脏、SSM和IFM样品，用于脂肪提取和磷脂分析。特别关注的是 心磷脂(CL)分析：CL含量、CL种类及CL氧化。 4.获取心脏组织用于分析酰基辅酶A和酰肉碱，特别强调丙二酰辅酶A是关键 燃料利用中的代谢物。 5.进行质谱分析，确定ETC的翻译后修饰和氧化修饰 亚基，尤其是复合体IV。 6.用透射电子显微镜对心脏组织和心脏SSM、IFM进行结构分析。 7.异前列腺素和氧化型低密度脂蛋白(OxLDL)定量作为体内氧化的一种测量 压力。 8.将对项目1和项目4的线粒体进行蓝色原生电泳法。 相关性(请参阅说明)： 线粒体是动力源，也是细胞。心力衰竭时，心脏会出现能量危机。 并与线粒体功能障碍有关。这一核心将提供线粒体与心脏的分离 狗和老鼠在实验条件下，并执行分析方法，以支持4个项目在 该计划项目拨款。

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.