13C-Metabolic Flux Analysis for Understanding Cardiac Energy Homeostasis

用于了解心脏能量稳态的 13C 代谢通量分析

• 批准号: 9327508
• 负责人: Scott B. Crown
• 金额: $ 5.92万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9327508
• 关键词: Acetyl Coenzyme A; Affinity; Attention; Bioenergetics; Biological Models; Branched-Chain Amino Acids; C57BL/6 Mouse; Carbohydrates; Carbon; Cardiac; Cardiac development; Cardiovascular system; Carnitine O-Acetyltransferase; Catabolism; Characteristics; Citric Acid Cycle; Computer Simulation; Data; Defect; Energy Metabolism; Experimental Designs; Fatty Acids; Fatty acid glycerol esters; Foundations; Future; Glucose; Goals; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Homeostasis; Intervention; Ketones; Knockout Mice; Label; Laboratories; Leucine; Link; Mass Spectrum Analysis; Measurement; Measures; Mechanics; Metabolic; Metabolism; Methodology; Methods; Mitochondria; Modeling; Mus; Nutrient; Oxides; Pathway interactions; Pattern; Perfusion; Pharmacology; Pharmacotherapy; Proteins; Pyruvate; Resolution; Role; Sample Size; Scientist; Site; Skeletal Muscle; Source; Stress; Systems Biology; Techniques; Testing; Therapeutic Intervention; Time; Tracer; Validation; Valine; Work; amino acid metabolism; base; carboxylation; cardiogenesis; design; experimental study; improved; in vivo; interest; mouse model; oxidation; pressure; propionyl-coenzyme A; simulation; therapeutic target; tool

Strong evidence links the development of cardiac hypertrophy and heart failure (HF) to dramatic alterations in mitochondrial fuel metabolism and bioenergetics. Specifically, the capacity of the heart to oxidize its chief fuels, fatty acids (FA) and glucose, becomes constrained, causing compensatory shifts in metabolism to alternative substrates. Recent work in the Muoio lab using mouse models and a system biology approach has identified marked perturbations in short chain carbon metabolites (acetyl CoA, ketones and branched chain amino acids (BCAA)-derived intermediates) as a strong signature of HF. Likewise, emerging findings from the Newgard lab link cardiometabolic stress to aberrant BCAA metabolism and inactivation of anaplerotic pathways that refill intermediates of the tricarboxylic acid cycle. Whereas static assessment of metabolite concentrations can highlight pathways deserving of further attention, measurement of metabolic flux is necessary to precisely pinpoint sites of metabolic dysregulation. Current heart perfusion methods to measure fluxes have several limitations including: 1) restriction to a single 13C-tracer per perfusion, despite various substrates of interest; 2) inadequate assessment of anaplerosis, as pyruvate is considered while other potentially important sources are ignored (e.g. BCAA-derived propionyl CoA); and 3) limited analysis of only a small fraction of the 13C-enrichment data generated in each experiment. To overcome these limitations, this project aims to develop and validate a powerful 13C-based “multiplex” MFA method, wherein several 13C- substrates (namely glucose, lactate, FA, and BCAA) can be applied to a single heart perfusion to evaluate substrate oxidation and anaplerotic fluxes, with emphasis on fluxes around the pyruvate, acetyl CoA and propionyl CoA nodes. This “multiplex” technique will reduce mouse sample size required for perfusions, while simultaneously increasing quality and quantity of flux information obtained. Lastly, this project will apply the “multiplex” MFA method to the carnitine acetyltransferase (CrAT) knockout mouse, which is a well-characterized model of altered acetyl and propionyl CoA metabolism. BCAA will serve as tool compounds to probe both acetyl CoA and propionyl CoA metabolism in CrAT- deficient hearts. Overall, this study will advance long-term goals of defining potential roles of CrAT and BCAA metabolism as factors that contribute to adaptive/maladaptive remodeling of the failing heart.

强有力的证据将心脏肥大和心力衰竭（HF）的发展与严重的 线粒体燃料代谢和生物能量学的改变。具体来说，心脏的能力 氧化它的主要燃料，脂肪酸（FA）和葡萄糖，变得受限，引起补偿性变化 在代谢到替代底物中。最近在Muoio实验室使用小鼠模型和系统的工作 生物学方法已经鉴定了短链碳代谢物（乙酰辅酶A， 酮和支链氨基酸（BCAA）衍生的中间体）作为HF的强特征。 同样，新的研究发现，从纽加德实验室链接心脏代谢的压力，异常支链氨基酸 补充三羧酸中间体的回补途径的代谢和失活 周期虽然代谢物浓度的静态评估可以突出值得研究的途径， 进一步注意，代谢通量的测量是必要的，以精确地定位代谢的位点。 失调目前测量流量的心脏灌注方法有几个局限性 包括：1）尽管有各种感兴趣的底物，但每次灌注仅限于单个13 C-示踪剂; 2） 回补的评估不足，因为丙酮酸被认为是，而其他潜在的重要 来源被忽略（例如，BCAA衍生的丙酰CoA）;以及3）仅对一小部分进行有限分析 每个实验中产生的13 C富集数据。为了克服这些局限性，该项目 旨在开发和验证一种强大的基于13 C的“多重”MFA方法，其中几个13 C- 底物（即葡萄糖、乳酸盐、FA和BCAA）可应用于单个心脏灌注， 评估底物氧化和回补通量，重点是丙酮酸盐周围的通量， 乙酰CoA和丙酰CoA节点。这种“多重”技术将减少小鼠样本量 这是灌注所需的，同时提高了所获得的通量信息的质量和数量。 最后，本研究将多重分子荧光分析方法应用于肉毒碱乙酰转移酶的研究 敲除小鼠，其是改变的乙酰基和丙酰CoA代谢的充分表征的模型。 支链氨基酸将作为工具化合物，探测CrAT中乙酰辅酶A和丙酰辅酶A的代谢。 心脏缺陷总的来说，这项研究将推进确定CrAT潜在作用的长期目标， BCAA代谢作为导致衰竭心脏适应性/适应不良重塑的因素。