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.
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