Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.

The myocardium responds to alterations in cardiac work by changing its rate of O2 consumption. This reflects an increase in the oxidative synthesis of ATP to meet the contractile demand for ATP. However, the biochemical mechanisms responsible for increased ATP synthesis are not fully understood. To localize the flux-controlling reaction(s) in the pathway of ATP synthesis, the effects of substrates and cardiac work on mitochondrial membrane potential (delta psi m), total tissue NADH-to-NAD+ ratio, and high-energy phosphate metabolites were examined in perfused rat hearts. Delta psi m was measured using the equilibrium distribution of tetraphenylphosphonium (33). Cytosolic phosphorylation potential, total tissue NADH-to-NAD+ ratio, and delta psi m were higher in hearts perfused with pyruvate than in those perfused with glucose. Increasing cardiac work induced a four-fold increase in O2 consumption, which was accompanied by 1) decreased or unaltered cytosolic ADP concentration, 2) increased tissue NADH-to-NAD+ ratio, and 3) decreased delta psi m. The results indicate that both NADH-generating reactions and the ATP synthase-catalyzed reaction are important in causing the increase in respiration that accompanies increased work. Because the activation of ATP synthase by cardiac work occurred in the absence of increases in delta psi m, ADP, and Pi, it is possible that the work-related acceleration in ATP synthesis may be due to modification of the kinetic properties of the ATP synthase.

To test how α-ketoglutarate dehydrogenase (α-KGDH) activity influences the balance between oxidative flux and transmitochondrial metabolite exchange, we monitored these rates in isolated mitochondria and in perfused rabbit hearts at an altered kinetics ( K m) of α-KGDH for α-ketoglutarate (α-KG). In isolated mitochondria, relative K mdropped from 0.23 mM at pH = 7.2 to 0.10 mM at pH 6.8 ( P < 0.05), and α-KG efflux decreased from 126 to 95 nmol ⋅ min−1 ⋅ mg−1. In intact hearts, K m was reduced with low intracellular pH, while matching control workload and respiratory rate with increased Ca2+(pHi = 7.20, perfusate CaCl2 = 1.5 mM; pHi = 6.89, perfusate CaCl2 = 3 ± 1 mM). Sequential13C nuclear magnetic resonance spectra from hearts oxidizing [2-13C]acetate provided tricarboxylic acid cycle flux and the exchange rate between α-KG and cytosolic glutamate ( F 1). Tricarboxylic acid cycle flux was 10 μmol ⋅ min−1 ⋅ g−1in both groups, but F 1 fell from a control of 9.3 ± 0.6 to 2.8 ± 0.4 μmol ⋅ min−1 ⋅ g−1at low K m. The results indicate that increased activity of α-KGDH occurs at the expense of α-KG efflux during support of normal workloads.