Mitochondrial Proteome Dynamics in Heart Failure Assessed with Heavy Water

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。 公共卫生相关性：心力衰竭困扰着600万美国人，尽管进行了积极的治疗，但患者的预后仍然很差。越来越多的证据表明，心脏衰竭的部分潜在问题是由于线粒体(细胞的一部分，将食物转化为心脏可以用来泵血的形式)导致的能量代谢缺陷。在这个项目中，我们将研究心力衰竭时线粒体功能障碍的潜在机制。

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. PUBLIC HEALTH RELEVANCE: Heart failure afflicts 6 million Americans, and despite aggressive treatment patient prognosis remains poor. There is growing evidence that part of the underlying problem in the failing heart is defective energy metabolism due to dysfunction mitochondria (the part of the cell that converts food to a form that the heart can use for pumping blood). In this project we will investigate the underlying mechanism for mitochondrial dysfunction in heart failure.