13C-Metabolic Flux Analysis for Understanding Cardiac Energy Homeostasis

强有力的证据表明，心肌肥厚和心力衰竭的发展与戏剧性 线粒体燃料代谢和生物能量学的改变。具体地说，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，导致补偿性变化 代谢成替代底物。Muoio实验室使用鼠标模型和一个系统的最新工作 生物学方法已经在短链碳代谢产物(乙酰辅酶A， 酮和支链氨基酸(BCAA衍生的中间体)是HF的强烈标志。 同样，纽加德实验室的新发现将心脏代谢应激与异常支链氨基酸联系在一起。 补充三元酸中间体的抗逆途径的代谢和失活 周而复始。而对代谢物浓度的静态评估可以突出值得 进一步注意，为了准确定位代谢部位，有必要测量代谢流量 监管失调。目前测量心脏血流流量的方法有几个局限性 包括：1)每次灌流仅限一种~(13)C示踪剂，尽管有多种感兴趣的底物； 对复苏的评估不充分，因为丙酮酸被认为是 来源被忽略(例如，支链氨基酸衍生的丙酰辅酶A)；以及3)仅对一小部分进行有限分析 在每一次实验中产生的13C浓缩数据。为了克服这些限制，这个项目 旨在开发和验证一种强大的基于13C的MFA方法，其中几种13C-MFA方法 底物(即葡萄糖、乳酸、FA和支链氨基酸)可用于单次心脏灌流 评估底物氧化和抗蚀通量，重点是丙酮酸周围的通量， 乙酰辅酶A和丙酰辅酶A节点。这种“多路传输”技术将减少小鼠的样本大小 灌流所需的流量，同时提高获得的流量信息的质量和数量。 最后，本项目将把“多重”MFA方法应用于肉碱乙酰转移酶(CrAT)。 基因敲除小鼠，这是一个很好地描述了乙酰基和丙酰辅酶A代谢改变的模型。 BCAA将作为工具化合物来探测CrAT中乙酰辅酶A和丙酰辅酶A的代谢。 有缺陷的心脏。总体而言，这项研究将推进界定CRAT和CRAT潜在作用的长期目标 支链氨基酸代谢是导致衰竭心脏适应性/非适应性重塑的因素。

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.