Researchers interested in metabolic changes in heart failure. Researchers using carbon-13 tracer analysis to measure metabolic flux. Over 5 million Americans have heart failure, with ~75 % of the cases characterized by antecedent hypertension (1). Metabolic changes during myocardial hypertrophy have been well studied, but substrate selection in heart failure has not been extensively probed (2). Citric acid cycle (CAC) flux is the primary source of reducing equivalents used for myocardial ATP production. Recently metabolic therapy putatively designed to increase ATP production in the heart has been combined with standard treatment paradigms to enhance care for patients with HF. The intent of this research was to more fully elucidate changes in myocardial substrate preference in HF and hopefully find pathways that could be modulated to improve myocardial function.All animal surgeries were approved by the UTSW IACUC. Briefly, C57Bl/6J mice 10-12 weeks in age were anesthetized and the aortic arch was accessed by a left lateral thoracotomy. The aorta was ligated over a 28 G needle, producing a discrete region of stenosis (3). Echocardiographic recordings revealed progressive deterioration of left ventricular systolic function. 21 days post-surgery the hearts (n=7) were excised and perfused in Langendorff mode at a constant perfusion pressure of 80 cm H₂O (4). Control hearts with a sham surgery were also analyzed (n=8). A Krebs-Henseleit buffer solution containing 8.2 mM [1,6-¹³C₂]glucose, 0.63 mM [U-¹³C]fatty acids (FAs), 0.17 mM [1,3-¹³C₂]acetoacetate, and 1 microunit/ml of insulin. The perfusate was bubbled with 95/5 O₂/CO₂ to maintain a pH of 7.4. After 30 minutes of perfusion the hearts were freeze clamped and extracted using perchloric acid. O₂ consumption was measured using a blood gas analyzer. The water soluble fraction was analyzed by ¹H decoupled, ¹³C NMR at 14.1 T using a Bruker 10 mm cryoprobe. The relative areas of the glutamate multiplets were measured using ACD NMR software. Fractional contributions of each substrate to acetyl-CoA production and anaplerosis were assessed by fitting the relative areas of the ¹³C peaks with tcaCALC. Figure 1 shows the labeling patterns in acetyl-CoA that are possible based on the perfusion conditions used for this study. Subsequent turns of the CAC will produce a distribution of the 32 possible ¹³C isotopomers of glutamate that can be modeled to produce estimates of substrate selection and absolute CAC flux (5). The HF model was not only significantly larger, but also consumed for oxygen per gram of tissue (Figure 2). We attribute increased O₂ consumption to increased work associated with contraction in the extremely fibrotic HF model. Carbon-13 spectra were acquired with excellent signal to noise for the sham surgery versus the HF model (Figure 3). The distribution of the ¹³C isotopomers as measured by NMR were fit to a model that included anaplerosis thru both Y_PC and Y_S (Figure 1). The relative contributions to acetyl-CoA production and flux through the anaplerotic pathways show that HF causes not only substrate switching, but also increased flux into the 4-carbon intermediate pools of the CAC (Figure 4). The preference in the failing heart for carbohydrates is increased as evidenced by the non-CHO versus CHO oxidation, an observation confirming many previous studies. The new, key observation is the significantly increased (P=0.025, 2-tail t-test) Y_s detected in the failing heart. Figure 5 (top) plots the absolute flux that can be inferred with the relative values of Figure 4 paired with the O₂ consumption. As can be seen, significant changes in absolute flux manifest for each of the substrates, as well as elevated Y_s flux. Figure 5 (bottom) renormalizes substrate competition as a fraction of acetyl-CoA production, showing that CHO oxidation is elevated, primarily at the expense of ketone oxidation. Metabolomic analysis of the CAC intermediate pool sizes (data not shown) indicates that the 4-carbon molecules (malate, fumarate, and succinate) are all lower in concentration in HF. It is likely that Y_s is activated in the failing heart as a mechanism for maintaining the pool sizes, allowing continued function of the CAC. Cataplerosis of these intermediates is commonly due to the need for amino acid synthesis.Carbon-13 isotopomer analysis is uniquely powerful for assessing substrate selection in functioning tissues. Using a well-accepted model of HF, we have determined that anaplerotic flux into the 4-carbon CAC intermediates (Y_s) is upregulated in HF. This phenomenon is likely related to increased autophagy (increased protein turnover) in the pathologically enlarged heart.