Mitochondrial Proteome Dynamics in Heart Failure Assessed with Heavy Water

用重水评估心力衰竭的线粒体蛋白质组动力学

• 批准号: 8519529
• 负责人: Takhar Kasumov
• 金额: $ 17.63万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8519529
• 关键词: Acceleration; Actins; American; Amino Acids; Animal Model; Animals; Body Water; Cardiac; Cardiac Myocytes; Cell Nucleus; Cells; Computer software; DNA; Data; Data Analyses; Deuterium; Deuterium Oxide; Development; Electron Transport; Energy Metabolism; Enzymes; Food; Functional disorder; Generations; Genetic Transcription; Grant; Half-Life; Heart; Heart Hypertrophy; Heart failure; Histones; Individual; Inner mitochondrial membrane; Label; Life; Liquid Chromatography; Location; Measurement; Measures; Metabolic; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Modeling; Myocardium; Myosin ATPase; Nuclear; Oral Administration; Oxidative Stress; Patients; Peptides; Plasma; Production; Protein Biosynthesis; Protein Dynamics; Proteins; Proteome; Proton-Translocating ATPases; Rattus; Reactive Oxygen Species; Resolution; Respiratory physiology; SLC2A1 gene; Sarcoplasmic Reticulum; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Techniques; Testing; Time; Tissues; Tracer; Water; blood pump; hexokinase; mitochondrial dysfunction; outcome forecast; oxidative damage; phospholamban; pressure; protein degradation; public health relevance; research study; stable isotope; tandem mass spectrometry

DESCRIPTION (provided by applicant): In this grant we will use a new stable isotope method called "proteome dynamics" to measure the rate of synthesis of individual mitochondrial proteins encoded on nuclear and mtDNA in the heart. Recent advances by our group in stable isotopic tracer methods and peptide analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables measurement of protein synthesis using deuterium (2H) labeled precursors and assessment of isotopically enriched protein products. Oral administration of "heavy water" (2H2O; a safe, non-radioactive compound) results in rapid steady state labeling of body water and transfer of 2H from 2H2O to 2H-labeled amino acids, which is followed by slow incorporation into proteins dependent upon the rate of synthesis of the specific protein. Assessment of protein dynamics requires measurement of enrichments of either tissue amino acids or total body water, and tryptic peptides, and calculation of the asymptotical number of 2H incorporated into a peptide using specialized software. We propose to use proteome dynamics to compare the synthesis rates for proteins encoded on mtDNA vs. nuclear DNA in the normal heart, in hearts with cardiac hypertrophy and early heart failure, and in hearts with advance heart failure and mitochondrial dysfunction. In addition, we will extend 2H2O tracers to measure the synthesis rate of mtDNA. Experiments will be performed in an established rat model of pressure overload HF that results in mitochondrial dysfunction. We will evaluate our hypotheses in three Specific Aims: 1) Determine if HF decreases the synthesis of proteins on the inner mitochondrial membrane encoded by mtDNA more than those on nuclear DNA. 2) Assess the synthesis rate of mtDNA by following the time course of the incorporation of 2H into mtDNA in the normal and failing heart. 3) Assess effects of HF on the synthesis rate of key cytosolic cardiac proteins.

描述(申请者提供)：在这项资助中，我们将使用一种名为蛋白质组动力学的新的稳定同位素方法来测量心脏中编码在细胞核和线粒体DNA上的单个线粒体蛋白质的合成速度。我们团队在稳定同位素示踪方法和液相色谱串联质谱(LC-MS/MS)多肽分析方面的最新进展，使我们能够使用氢(2H)标记的前体来测量蛋白质合成，并评估同位素浓缩的蛋白质产品。口服“重水”(2H2O；一种安全的非放射性化合物)可以快速稳定地标记体内的水，并将2H从2H2O转移到2H标记的氨基酸中，然后根据特定蛋白质的合成速度缓慢地结合到蛋白质中。对蛋白质动力学的评估需要测量组织氨基酸或全身水分和胰蛋白酶多肽的丰度，并使用专门的软件计算结合到多肽中的2H的渐近数。我们建议使用蛋白质组动力学来比较正常心脏、心肌肥厚和早期心力衰竭以及晚期心力衰竭和线粒体功能障碍心脏中线粒体DNA上编码的蛋白质的合成率。此外，我们将扩展2H2O示踪剂来测量mtDNA的合成速率。实验将在已建立的压力超负荷HF导致线粒体功能障碍的大鼠模型中进行。我们将在三个具体目标上评估我们的假设：1)确定HF是否比核DNA更能减少线粒体内膜上编码的蛋白质的合成。2)通过观察正常心脏和衰竭心脏线粒体DNA中2H掺入的时间进程，评估线粒体DNA的合成率。3)评价HF对心肌细胞内关键蛋白合成速率的影响。