To maintain ATP constant in the cell, mitochondria must sense cellular ATP utilization and transduce this demand to F 0 -F 1 -ATPase. In spite of a considerable research effort over the past three decades, no combination of signal(s) and kinetic function has emerged with the power to explain ATP homeostasis in all mammalian cells. We studied this signal transduction problem in intact human muscle using 31 P NMR spectroscopy. We find that the apparent kinetic order of the transduction function of the signal cytosolic ADP concentration ([ADP]) is at least second order and not first order as has been assumed. We show that amplified mitochondrial sensitivity to cytosolic [ADP] harmonizes with in vitro kinetics of [ADP] stimulation of respiration and explains ATP homeostasis also in mouse liver and canine heart. This result may well be generalizable to all mammalian cells.Prior work considered that mitochondria behave as a transducer with approximately first order response characteristics (1-4). This means that the response of mitochondrial oxidative phosphorylation (MOP) 1 to a stimulus would follow an approximately hyperbolic relation according to a Michaelis-Menten mechanism for the signal transduction (2, 3). With this understanding, the hypothesis that mitochondria detect variations in ATP utilization simply by sensing the variation in cytosolic [ADP] (2, 3) had to be discarded as a general mechanism after studies of the in situ dog heart showed 2-fold increases in MOP flux without much change in [ADP] (4). These observations led to consideration of alternative signals but not alternative kinetic functions of ADP-mediated signal transduction (1, 4). This was unfortunate, because earlier work on isolated mitochondria had shown that the response of MOP to changes in [ADP] is not hyperbolic (5, 6). Therefore, it remains possible that a higher order kinetic function for extramitochondrial [ADP] stimulation of MOP is responsible for the maintenance of energy balance in the mammalian cell.Here, we studied cytosolic [ADP] transduction in an intact cellular system. We used a general and unbiased analysis to test the apparent kinetic order of the transduction function. The generality of the in vivo result is tested against published kinetics of ADP stimulation of MOP in various other systems, and its implications for understanding the biochemistry of mitochondria and the integrative physiology of mitochondrial function in the cell are discussed. MATERIALS AND METHODS 31P NMR Spectroscopy of Intact Muscle-Phosphocreatine (PCr), P i , and ATP 31 P NMR resonances in well perfused human forearm flexor muscle of six consenting, healthy adult volunteers (five males and one female; age, 28 -55 years) were measured using high time resolution (7 s) 31 P NMR spectroscopy, and data acquisition and analysis methods developed in this laboratory (7, 8). 31P NMR spectra were collected using a CSI spectrometer operating at 2 tesla (General Electric). Different energy balance states were imposed by supramaximal percutaneous nerve stimu...

MR is a powerful technique for studying the biomechanical and functional properties of skeletal muscle in vivo in health and disease. This review focuses on 31 P, 1 H and 13 C MR spectroscopy for assessment of the dynamics of muscle metabolism and on dynamic 1 H MRI methods for non-invasive measurement of the biomechanical and functional properties of skeletal muscle. The information thus obtained ranges from the microscopic level of the metabolism of the myocyte to the macroscopic level of the contractile function of muscle complexes. The MR technology presented plays a vital role in achieving a better understanding of many basic aspects of muscle function, including the regulation of mitochondrial activity and the intricate interplay between muscle fiber organization and contractile function. In addition, these tools are increasingly being employed to establish novel diagnostic procedures as well as to monitor the effects of therapeutic and lifestyle interventions for muscle disorders that have an increasing impact in modern society.

Data from 31 P-nuclear magnetic resonance spectroscopy of human forearm flexor muscle were analyzed based on a previously developed model of mitochondrial oxidative phosphorylation (PLoS Comp Bio 1: e36, 2005) to test the hypothesis that substrate level (concentrations of ADP and inorganic phosphate) represents the primary signal governing the rate of mitochondrial ATP synthesis and maintaining the cellular ATP hydrolysis potential in skeletal muscle. Model-based predictions of cytoplasmic concentrations of phosphate metabolites (ATP, ADP, and Pi) matched data obtained from 20 healthy volunteers and indicated that as work rate is varied from rest to submaximal exercise commensurate increases in the rate of mitochondrial ATP synthesis are effected by changes in concentrations of available ADP and Pi. Additional data from patients with a defect of complex I of the respiratory chain and a patient with a deficiency in the mitochondrial adenine nucleotide translocase were also predicted the by the model by making the appropriate adjustments to the activities of the affected proteins associates with the defects, providing both further validation of the biophysical model of the control of oxidative phosphorylation and insight into the impact of these diseases on the ability of the cell to maintain its energetic state. computational model; mitochondria; cellular energetics; oxidative phosphorylation; 31 P-NMR spectroscopy MITOCHONDRIAL OXIDATIVE ADP phosphorylation is the primary source of ATP in skeletal muscle during aerobic exercise. Thus, to maintain the free energy state of the cytoplasmic phosphoenergetic compounds ATP, ADP, and P i , oxidative phosphorylation is modulated to match the rate of ATP utilization during exercise. It has recently been shown through computational model-based analysis of data obtained from 31 P-NMR spectroscopy of working in vivo dog hearts that the primary control mechanism operating in cardiomyocytes is feedback of substrate concentrations for ATP synthesis (5). In other words, changes in the concentrations of the products generated by the utilization of ATP in the cell, ADP and P i , effect changes in the rate at which mitochondria utilize those products to resynthesize ATP (5).Here the question of whether this same mechanism can explain the observed data on the control of oxidative metabolism in skeletal muscle is investigated. Previous analyses of 31 P-NMR spectroscopy ( 31 P-MRS) data on energy balance in exercising skeletal muscle have mainly focused on testing ADP feedback control of mitochondrial ATP synthesis using black box descriptions of the mitochondrial ATP synthetic pathway (8, 14 -16, 28), P i acceptor control (7), and thermodynamic control involving quasi-linear relations between cytoplasmic Gibbs free energy of ATP hydrolysis and mitochondrial ATP synthesis flux (13,18, 31). Yet, to date, these 31 P-MRS data have not been adequately explained based on a detailed mechanistic model of oxidative phosphorylation and cellular energetics.To analyze and interpret data from s...

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...

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