NMR OF MITOCHONDRIAL TRANSPORTERS IN CARDIAC HYPERTROPHY

这项建议使用核磁共振在跨学科方法中的新应用，以表征和干预肥厚和衰竭心脏中受损的能量学的分子基础。在肥大中，表型重编程发生，在发育早期表达的基因被重新表达。我们的数据显示，这种趋势延伸到线粒体膜上的转运体，通过中间能量产生来调节通量。转运体是α-酮戊二酸(AKG)-苹果酸交换器和肉碱棕榈酰基转移酶I(CPT1)。我们实验室令人兴奋的发现表明，13C核磁共振不仅对代谢流量敏感，而且对完整心脏的这些线粒体转运蛋白也敏感。同样令人兴奋的是，我们发现了基因剪接形式在心脏中的表达，这种形式编码CPT1的异构体，它将长链脂肪酸携带到线粒体中进行氧化。这些结果，再加上我们在活体大鼠心脏中成功的腺病毒基因表达结果，导致了对转运蛋白表达和功能的全面分析。我们的目的是检测和干预心脏基因表达的重新编程，以阐明分子水平的代谢流量调节作为失代偿性肥厚能量受损的基础。该提案测试了三个假设：1)肥厚时基因表达逆转为糖酵解代谢增加和脂肪酸使用减少，其显著特征是：a)AKG-苹果酸交换器的表达和活性增加；b)CPT1亚型与mRNA剪接变异体的活性不同；2)CPT1亚型的变化降低肥厚心脏的能量潜力；3)这些蛋白质变化发生在人类心力衰竭中，动物研究为其提供了功能意义。最初，我们将分离的大鼠心脏转运蛋白速率和代谢流量的13C核磁共振测量与传统的分子方法相结合来评估重新编程。其次，我们用~(13)C核磁共振来评估腺病毒基因转移对CPT1亚型的干预作用。使用心脏肥厚的主动脉带模型，我们建议：1)表征正常心脏、肥厚心脏(12周带)和衰竭心脏(20周)的调节转运速率；以及2)通过过度表达本来减少的CPT1亚型来干预肥厚心脏受损的能量。随后，研究扩展到对转运蛋白在人类心肌中表达的相关评估。因此，我们对心肌肥厚的核磁共振研究提出了一个新的研究水平。这些方案将从动物模型中建立对患病人类心肌异构体变化的功能理解。长期目标是建立13C核磁共振来评估体内代谢流量调节的分子变化，这些变化有助于从正常心肌向衰竭心肌的转变。

This proposal employs novel applications of NMR in an interdisciplinary approach to characterize and intervene in the molecular basis of impaired energetics in hypertrophied and failing hearts. In hypertrophy, a phenotypic reprogramming occurs whereby genes expressed early in development are re-expressed. Our data show that this trend extends toward transporters on the mitochondrial membrane that regulate flux through intermediary energy production. The transporters are alpha-ketoglutarate (aKG)-malate exchanger and carnitine palmitoyltransferase I (CPT1). Exciting findings from our laboratory show 13C NMR is sensitive not only to metabolic flux, but also to these mitochondrial transporters in intact hearts. Equally exciting is our new evidence for cardiac expression of gene splicing forms, encoding isoforms of CPT1 which carries long chain fatty acids into mitochondria for oxidation. These results, coupled with our successful adenoviral gene expression results in in vivo rat hearts, lead to this comprehensive analysis of transporter expression and function. Our objective is to examine and intervene in reprogramming cardiac gene expression to elucidate molecular-level, metabolic flux regulation as the basis for impaired energetics in decompensated hypertrophy. The proposal tests three hypotheses that: 1) reversion of gene expression in hypertrophy to increased glycolytic metabolism and reduced fatty acid use, is marked by: a) increased expression and activity of aKG-malate exchangers; b) differential activities of CPT1 isoforms from mRNA splicing variants; 2) isoform changes in CPT1 reduce energy potential in hypertrophied hearts; and 3) these protein changes occur in human heart failure for which animal studies provide functional implications. Initially, we combine 13C NMR measures of transporter rates and metabolic flux in isolated rat hearts with traditional molecular methods to assess reprogramming. Secondly, we use 13C NMR to assess adenoviral gene transfer intervention on CPT1 isoforms. Using the aortic-banding model of cardiac hypertrophy we propose to: 1) characterize regulatory transport rates in normal hearts, hypertrophied hearts (12 weeks banding), and at failure (20 weeks); and 2) intervene on the impaired energetics of hypertrophied hearts by overexpressing otherwise reduced CPT1 isoforms. Investigation then extends to correlative evaluations of transporter expression in human myocardium. Thus, we propose a new level of investigation for NMR studies of cardiac hypertrophy. The protocols will establish the functional understanding from animal models for isoform changes in diseased human myocardium. The long range goal is to establish 13C NMR to assess molecular changes in metabolic flux regulation in vivo that contribute to the transition from normal to failing myocardium.