Mitochondrial Proteome Dynamics in Heart Failure Assessed with Heavy Water

描述（由申请人提供）：在这项资助中，我们将使用一种称为“蛋白质组动力学”的新的稳定同位素方法来测量心脏中核和mtDNA上编码的单个线粒体蛋白质的合成速率。本小组在稳定同位素示踪方法和液相色谱-串联质谱（LC-MS/MS）肽分析方面的最新进展，使使用氘（2H）标记前体测量蛋白质合成和评估同位素富集的蛋白质产物成为可能。口服“重水”（2H2O，一种安全的非放射性化合物）可快速稳定地标记体内水分，并将2H从2H2O转移到2H标记的氨基酸中，随后根据特定蛋白质的合成速度缓慢地与蛋白质结合。蛋白质动力学的评估需要测量组织氨基酸或全身水分和色氨酸的富集程度，并使用专门的软件计算纳入肽的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的心脏以及晚期心力衰竭和线粒体功能障碍的心脏中mtDNA与核DNA编码的蛋白质合成率。此外，我们将扩展2H2O示踪剂来测量mtDNA的合成速率。实验将在导致线粒体功能障碍的压力过载HF大鼠模型中进行。我们将在三个具体目标中评估我们的假设：1)确定HF是否比核DNA更能减少mtDNA编码的线粒体内膜上蛋白质的合成。2)通过观察正常和衰竭心脏中2H掺入mtDNA的时间过程来评估mtDNA的合成速率。3)评估HF对心脏关键细胞质蛋白合成速率的影响。

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.