NMR OF MITOCHONDRIAL TRANSPORTERS IN CARDIAC HYPERTROPHY

该建议采用跨学科方法的NMR新应用来表征和干预肥厚和衰竭心脏中受损能量学的分子基础。在肥厚中，表型重编程发生，即在发育早期表达的基因被重新表达。我们的数据表明，这种趋势延伸到线粒体膜上的转运体，通过中间能量生产调节通量。转运体是α -酮戊二酸(aKG)-苹果酸交换剂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通量敏感，而且对完整心脏中的这些线粒体转运蛋白也敏感。同样令人兴奋的是我们关于心脏基因剪接形式表达的新证据，编码CPT1的同种异构体，CPT1携带长链脂肪酸进入线粒体氧化。这些结果，加上我们在体内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。我们的目的是检查和干预心脏基因表达的重编程，以阐明分子水平上代谢通量调节作为失代偿肥厚中能量受损的基础。该提案验证了三个假设：1)肥大的基因表达逆转为糖酵解代谢增加和脂肪酸使用减少，其标志是：a) akg -苹果酸交换物的表达和活性增加；b) mRNA剪接变异体CPT1亚型的差异活性；2) CPT1异构体的改变降低了肥厚心脏的能量势；3)这些蛋白质变化发生在人类心力衰竭中，动物研究提供了功能意义。最初，我们将13C核磁共振测量的转运蛋白率和代谢通量与传统的分子方法结合起来评估重编程。其次，我们使用13C核磁共振来评估对CPT1亚型的腺病毒基因转移干预。利用心肌肥厚的主动脉束带模型，我们建议：1)表征正常心脏、肥厚心脏（束带12周）和衰竭心脏（束带20周）的调节转运率；2)通过过表达或减少CPT1亚型来干预肥厚心脏的能量学受损。然后研究扩展到转运蛋白在人心肌中的表达的相关评价。因此，我们为心脏肥厚的核磁共振研究提出了一个新的研究水平。该方案将从动物模型中建立对患病人类心肌同种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通量调节的分子变化，这些变化有助于心肌从正常到衰竭的转变。

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.