The transduction function for ADP stimulation of mitochondrial ATP synthesis in skeletal muscle was reconstructed in vivo and in silico to investigate the magnitude and origin of mitochondrial sensitivity to cytoplasmic ADP concentration changes. Dynamic in vivo measurements of human leg muscle phosphocreatine (PCr) content during metabolic recovery from contractions were performed by 31 P-NMR spectroscopy. The cytoplasmic ADP concentration ([ADP]) and rate of oxidative ATP synthesis (Jp) at each time point were calculated from creatine kinase equilibrium and the derivative of a monoexponential fit to the PCr recovery data, respectively. Reconstructed [ADP]-Jp relations for individual muscles containing more than 100 data points were kinetically characterized by nonlinear curve fitting yielding an apparent kinetic order and ADP affinity of 1.9 Ϯ 0.2 and 0.022 Ϯ 0.003 mM, respectively (means Ϯ SD; n ϭ 6). Next, in silico [ADP]-Jp relations for skeletal muscle were generated using a computational model of muscle oxidative ATP metabolism whereby model parameters corresponding to mitochondrial enzymes were randomly changed by 50 -150% to determine control of mitochondrial ADP sensitivity. The multiparametric sensitivity analysis showed that mitochondrial ADP ultrasensitivity is an emergent property of the integrated mitochondrial enzyme network controlled primarily by kinetic properties of the adenine nucleotide translocator. mitochondria; nuclear magnetic resonance; mathematical modeling; regulation; adenosine 5Ј-diphosphate THE METABOLIC REGULATION UNDERLYING ENERGY BALANCE in mammalian cells has long been the subject of investigation, in particular regulation of mitochondrial ATP synthesis (1). At first, a relatively straightforward picture emerged from studies in isolated mitochondria; a feedback control loop involving transduction of changes in the extramitochondrial concentrations of the ATP hydrolysis products ADP and P i to the intramitochondrial ATP synthetic network during cellular work explained energy balance (10, 13).31 P-NMR spectroscopy (11) and computational modeling (28) later showed that mitochondrial sensing of concentration changes in ADP alone sufficed to explain energy balance in skeletal muscle. However, in cardiac muscle, near-constant phosphocreatine (PCr), and thereby ADP, concentrations were measured during work jumps (3). This observation led to the proposition of a second, if not alternative, mitochondrial metabolic control mechanism in excitable cells such as cardiac muscle, i.e., a feedforward control loop involving direct or indirect transduction of intracellular calcium concentration changes to the mitochondrial ATP synthetic network (1). More recently, yet another alternative respiratory control mechanism of particular relevance to energy balance in the heart has been identified (2). It involves a mix of feedforward and feedback kinetic effects of P i on multiple reactions in the oxidative ADP phosphorylation pathway (7).What has been relatively lacking for each of the postulated re...

An MR-compatible ergometer was developed for in-magnet whole-body human exercise testing. Designed on the basis of conventional mechanically braked bicycle ergometers and constructed from nonferrous materials, the ergometer was implemented on a 1.5-T whole-body MR scanner. A spectrometer interface was constructed using standard scanner hardware, complemented with custom-built parts and software to enable gated data acquisition during exercise. High-quality 31 P NMR spectra were reproducibly obtained from the medial head of the quadriceps muscle of the right leg of eight healthy subjects during twolegged high-frequency pedaling (80 revolutions per minute) at three incremental workloads, including maximal. Muscle phosphocreatine content dropped 82%, from 32.2 6 1.0 mM at rest to 5.7 6 1.1 mM at maximal workload (mean 6 standard error; n 5 8), indicating that the majority of quadriceps motor units were recruited. The cardiovascular load of the exercise was likewise significant, as evidenced by heart rates of 150 (610%) beats per minute, measured immediately afterward. As such, the newly developed MR bicycling exercise equipment offers a powerful new tool for clinical musculoskeletal and cardiovascular MR investigation. The basic design of the ergometer is highly generic and adaptable for application on a wide selection of whole-body MR scanners. Magn Reson Med 63:257-261,

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The regulation of the 100-fold dynamic range of mitochondrial ATP synthesis flux in skeletal muscle was investigated. Hypotheses of key control mechanisms were included in a biophysical model of oxidative phosphorylation and tested against metabolite dynamics recorded by 31 P nuclear magnetic resonance spectroscopy ( 31 P MRS). Simulations of the initial model featuring only ADP and Pi feedback control of flux failed in reproducing the experimentally sampled relation between myoplasmic free energy of ATP hydrolysis (ΔG p = ΔG p o′ +RT ln ([ADP][Pi]/[ATP]) and the rate of mitochondrial ATP synthesis at low fluxes (<0.2 mM/s). Model analyses including Monte Carlo simulation approaches and metabolic control analysis (MCA) showed that this problem could not be amended by model re-parameterization, but instead required reformulation of ADP and Pi feedback control or introduction of additional control mechanisms (feed forward activation), specifically at respiratory Complex III. Both hypotheses were implemented and tested against time course data of phosphocreatine (PCr), Pi and ATP dynamics during post-exercise recovery and validation data obtained by 31 P MRS of sedentary subjects and track athletes. The results rejected the hypothesis of regulation by feed forward activation. Instead, it was concluded that feedback control of respiratory chain complexes by inorganic phosphate is essential to explain the regulation of mitochondrial ATP synthesis flux in skeletal muscle throughout its full dynamic range.

The longstanding problem of rapid inactivation of the glycolytic pathway in skeletal muscle after contraction was investigated using 31 P NMR spectroscopy and computational modelling. Accumulation of phosphorylated glycolytic intermediates (hexose monophosphates) during cyclic contraction and subsequent turnover during metabolic recovery was measured in vivo in human quadriceps muscle using dynamic 31 P NMR spectroscopy. The concentration of hexose monophosphates in muscle peaked 40 s into metabolic recovery from maximal contractile work at 6.9 ± 1.3 mm (mean ± s.d.; n = 8) and subsequently declined at a rate of 0.009 ± 0.001 mm s −1 . It was next tested whether the current knowledge of the kinetic controls in the glycolytic pathway in muscle integrated in the Lambeth and Kushmerick computational model of skeletal muscle glycolysis explained the experimental data. It was found that the model underestimated the magnitude of deactivation of the glycolytic pathway in resting muscle, resulting in depletion of glycolytic intermediates and substrate for oxidative ATP synthesis. Numerical analysis of the model identified phosphofructokinase and pyruvate kinase as the kinetic control sites involved in deactivation of the glycolytic pathway. Ancillary 100-fold inhibition of both phosphofructokinase and pyruvate kinase was found necessary to predict glycolytic intermediate and ADP concentrations correctly in resting human muscle. Incorporation of this information into the model resulted in highly improved agreement between predicted and measured in vivo dynamics of hexose monophosphates in muscle following contraction. We concluded that silencing of the glycolytic pathway in muscle following contraction is most likely to be mediated by phosphofructokinase and pyruvate kinase inactivation on a time scale of seconds and minutes, respectively, and is necessary to prevent depletion of vital cellular substrates.