NMR of Mitochondrial Transporters in Cardiac Hypertrophy

描述(由申请人提供)：拟议研究的总体目标是阐明和应对因替代途径的代偿招募和压力超负荷心脏代谢基因表达改变而导致的脂肪酸和碳水化合物代谢产生能量的低效。AIMS旨在产生新的代谢策略，以减轻导致明显心力衰竭的心肌病的发展。正在进行的工作已阐明：1)依赖于NADPH的苹果酸酶(ME)在心肌肥厚中的表达增加，从而增加了进入TCA周期第二个周期的促排斥量，并可能影响脂肪酸氧化和储存的调节，对收缩性能有明显的影响；2)肝脏(L)肉碱棕榈酰转移酶I亚型(L-CPT1)在肥厚心脏中的表达增加，与脂肪酸氧化减少有关；3)在肥厚心脏中，三酰甘油(TAG)池的含量和周转率均大幅下降，与TAG对脂肪酸氧化的贡献减少一致。基于当前资金期的这些关键发现，实验验证了这样的假设：维持基线脂肪酸氧化对脂肪酸氧化的贡献需要正常的TAG含量和周转率，这对收缩能力的代谢支持的灵活性至关重要，以及心肌肥厚中脂质储存动力学的异常调节，部分原因是通过苹果酸酶的逆转和通过L-CPT1的脂肪酸氧化的不适应转变，影响收缩的代谢效率。这一假说将使用转基因大鼠心脏和压力超负荷肥厚转基因小鼠心脏的动态模式13C核磁共振进行验证。目的1用腺病毒介导的正常大鼠心肌ME高表达和肥厚大鼠ME的RNA抑制，研究ME高表达对肥厚大鼠心脏甘油三酯动力学和氧化还原调节的影响。这一目的还测试了脂肪酸链长度和膳食脂肪对心肌肥厚中TAG动力学的影响，以及由于ME表达的差异而导致的丙酮酸的代谢命运。目的2通过抑制L CPT1RNA表达，探讨L CPT1RNA抑制对肥厚大鼠心脏脂肪酸储存动力学与氧化相互作用及平衡的影响。目的3探讨脂肪酸转运蛋白1(FATP1)过表达和PPAR1低表达转基因小鼠心肌组织中脂肪酸摄取增加和PPAR1表达增加对心肌TAG动力学和氧化的影响。我们不是研究单一的酶，而是提出一种综合的方法来研究心肌肥厚时代谢酶表达的潜在不适应性变化，同时探索肥厚过程中TAG动力学降低的潜在机制和功能意义。这些预期的发现将有助于对一个临床上重要的局部问题有一个基本的、机械性的理解，这个问题是在失代偿性肥厚的发病机制中心脏脂质动力学失调和收缩功能障碍之间的联系。 与公共健康相关：这项研究的具体重点是心脏改变脂肪酸氧化和储存的机制，这些机制与肥厚心脏中脂肪酸氧化酶和碳水化合物代谢的替代途径不明地上调有关，这些都是PI在上一个资助期间首次确定的。重要的是，我们的发现表明，在心肌肥厚的实验模型中，这些代谢变化似乎是可逆的，直接影响心脏的收缩功能。在压力超负荷性肥厚的大鼠模型和外源性基因输送的大鼠心脏以及增强的心脏脂肪酸摄取和代谢的转基因小鼠模型中，将研究心肌脂质含量和动力学与压力超负荷性肥厚的心功能障碍的关系。我们将通过核磁共振和改进的心脏基因传递方案，应用对完整、跳动的大鼠心脏代谢流量的新观察，来监测和干预肥厚心脏产生能量的代谢途径中发生的潜在的适应不良的变化的发展。因此，本研究旨在阐明肥厚心脏脂代谢受损的机制，这些机制直接与心功能受损和失代偿性肥厚的发展有关，并测试导致这种功能障碍的代谢过程的潜在治疗干预。

DESCRIPTION (provided by applicant): The overall objective of the proposed research is to elucidate and counter inefficiencies in fatty acid and carbohydrate metabolism for energy production, imposed by compensatory recruitment of alternative pathways and altered metabolic gene expression in pressure overloaded hearts. Aims are intended to yield new metabolic strategies to mitigate the development of cardiomyopathy leading to overt heart failure. Ongoing work has elucidated 1) increased NADPH-dependent malic enzyme (ME) expression in cardiac hypertrophy that increases anaplerotic flux into the second span of the TCA cycle and may impact on regulation of fatty acid oxidation and storage, with apparent effects on contractility; 2) increased expression of the liver (L) isoform of carnitine palmitoyltransferase I (L-CPT1) in hypertrophied hearts that coincides with reduced fatty acid oxidation; 3) large reductions in both content and turnover rates of the triacylglyceride (TAG) pool in hypertrophied hearts that coincide with a loss of the contribution from TAG to fatty acid oxidation. Based on these key findings of the current funding period, experiments test the hypothesis that normal TAG content and turnover are required to maintain baseline lipolytic contributions to fatty acid oxidation, which is critical to the flexibility of the metabolic support of contractility, and that dysregulation of lipid storage dynamics in cardiac hypertrophy, in part, due to maladaptive shifts in anaplerosis via malic enzyme and fatty acid oxidation via L- CPT1, affects the metabolic efficiency of contraction. The hypothesis will be tested using dynamic-mode 13C NMR of genetically altered rat hearts and transgenic mouse hearts with pressure overload hypertrophy. Aim 1 investigates the effects of increased ME expression on both triglyceride dynamics and redox regulation in hypertrophied rat hearts using adenoviral-based ME overexpression in normal hearts and RNA suppression of ME in hypertrophy. This aim also tests the effects of fatty acid chain length and dietary fat on TAG dynamics in cardiac hypertrophy and the metabolic fate of pyruvate due to differential ME expression. Aim 2 tests the functional significance of increased L-CPT1 expression on the reciprocal activity and balance between fatty acid storage kinetics and oxidation in hypertrophied rat hearts using L-CPT1 RNA inhibition. Aim 3 explores the influences of augmented fatty acid uptake and PPAR1 expression on the changes in TAG dynamics and oxidation in hypertrophied hearts of transgenic mice with either fatty acid transporter 1 (FATP1) overexpression or low overexpression of PPAR1. Rather than investigate a single enzyme, we propose an integrative approach to investigate potentially maladaptive changes in metabolic enzyme expression in cardiac hypertrophy, while exploring the potential mechanisms for, and functional significance of reduced TAG dynamics in hypertrophy. The anticipated findings will contribute a basic, mechanistic understanding to a topical problem of clinical importance, which is the link between dysregulation of cardiac lipid dynamics and contractile dysfunction in the pathogenesis of decompensated hypertrophy. PUBLIC HEALTH RELEVANCE: The specific focus of this research are mechanisms of altered fatty acid oxidation and storage by the heart, that are linked to unexplained upregulation of enzymes for fatty acid oxidation and alternative pathways of carbohydrate metabolism in the hypertrophied heart, that were first identified by the PI during the previous funding period. Importantly, our findings indicate that these metabolic changes appear to be reversible in experimental models of cardiac hypertrophy with direct effects on the contractile function of the heart. The relationship of myocardial lipid content and dynamics to cardiac dysfunction in pressure overload hypertrophy will be examined in isolated hearts from rat models of pressure overload hypertrophy and exogenous gene delivery and transgenic mouse models of augmented, cardiac fatty acid uptake and metabolism. We will apply novel observations of metabolic flux in the intact, beating rat heart via NMR and improved cardiac gene delivery schemes, to both monitor and intervene in the development of potentially maladaptive, alterations that occur in the energy yielding, metabolic pathways of the hypertrophied heart. Therefore, this research is intended to elucidate mechanisms of impaired lipid metabolism in hypertrophied hearts that directly related to impaired cardiac function and the development of decompensated hypertrophy, and to test potentially therapeutic interventions of the metabolic processes contributing to such dysfunction.