Issues of exploration: human health and wellbeing during a mission to Mars.

Today, the tools are in our hands to enable us to travel away from our home planet and become citizens of the solar system. Even now, we are seriously beginning to develop the robust infrastructure that will make the 21st century the Century of Space Travel. But this bold step must be taken with due concern for the health, safety and wellbeing of future space explorers. Our long experience with space biomedical research convinces us that, if we are to deal effectively with the medical and biomedical issues of exploration, then dramatic and bold steps are also necessary in this field. We can no longer treat the human body as if it were composed of muscles, bones, heart and brain acting independently. Instead, we must lead the effort to develop a fully integrated view of the body, with all parts connected and fully interacting in a realistic way. This paper will present the status of current (2000) plans by the National Space Biomedical Research Institute to initiate research in this area of integrative physiology and medicine. Specifically, three example projects are discussed as potential stepping stones towards the ultimate goal of producing a digital human. These projects relate to developing a functional model of the human musculoskeletal system and the heart.

Three dimensional ultrasound analysis of fascicle orientation in human tibialis anterior muscle enables analysis of macroscopic torque at the cellular level.

The purpose of this study was to test the hypothesis that the internal structure of the bipennate human tibialis anterior muscle is sufficiently homogenous throughout the muscle that the cellular stresses could be interpreted correctly from measurable anatomic properties and torque in the limb. This result is needed for facile comparison of extrinsic mechanical data and intrinsic energetic fluxes. Three-dimensional imaging of the fascicles of the human tibialis anterior muscle was made by capturing a series of ultrasound images while registering their location in space. Subsequent tracing of hundreds of structures in the ultrasound images with the use of custom software identified muscle boundaries, tendon surfaces, and fascicles as anatomic elements in 3-D space. The tendon was reconstructed as a mesh through the tracings identified as a component of the tendon. The angle of insertion of each identified fascicle at the tendon was calculated against the nearest normal in the mesh of the tendon. In three subjects the average angle of insertion of the fascicles onto the internal tendon was 11 degrees (coefficient of variation 40%). The angle decreased along the length of the muscle from approximately 15 degrees near the belly of the muscle to 6 degrees near the ankle in fascicles superior and inferior to the central tendon. The angle increased by several degrees during a voluntary contraction. Despite the differences in angles of insertion that can be measured, these distinctions have little significance for the distribution of forces along cellular axes within the muscle: the angles, their distribution within the muscle and change with contraction are small. For this bipennate muscle the cosine of the angle of insertion of the cellular bundles is always close to unity. Thus measurements of whole muscle mechanical data are simply related to mechanical stress of its cells.

Energetics of muscle contraction: the whole is less than the sum of its parts.

Understanding muscle energetics is a problem in optimizing supply of ATP to the demands of ATPases. The complexity of reactions and their fluxes to achieve this balance is greatly reduced by recognizing constraints imposed by the integration of common metabolites at fixed stoichiometry among modular units. ATPase is driven externally. Oxidative phosphorylation and glycogenolysis are the suppliers. We focus on their regulation which involves different controls, but reduces to two principles that enable facile experimental analysis of the supply and demand fluxes. The ratio of concentration of phosphocreatine (PCr) to ATP, not their individual values, sets the range of achievable concentrations of ADP in resting and active muscle (at fixed pH) in different cell types. This principle defines the fraction of available flux of oxidative phosphorylation utilized (at fixed enzyme activities). Then the kinetics of PCr recovery defines the kinetics of oxygen supply and substrate utilization. The second principle is the constancy of PCr and H(+) (lactate) production by glycogenolysis due to the coupling of ATPase and glycolysis. This principle enables glycogenolytic flux to be measured from intracellular proton loads. Further simplification occurs because the magnitude of the interacting fluxes and metabolite concentrations are specified within narrow limits when both the resting and active fluxes are quantified. Thus there is a small set of rules for assessing and understanding the thermodynamics and kinetics of muscle energetics.

A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle.

A system analysis of ATP free energy metabolism in skeletal muscle was made using the principles of metabolic control theory. We developed a network model of ATP free energy metabolism in muscle consisting of actomyosin ATPase, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, and mitochondria. These components were sufficient to capture the major aspects of the regulation of the cytosolic ATP-to-ADP concentration ratio (ATP/ADP) in muscle contraction and had inherent homeostatic properties regulating this free energy potential. As input for the analysis, we used ATP metabolic flux and the cytosolic ATP/ADP at steady state at six contraction frequencies between 0 and 2 Hz measured in human forearm flexor muscle by (31)P-NMR spectroscopy. We used the mathematical formalism of metabolic control theory to analyze the distribution of fractional kinetic control of ATPase flux and the ATP/ADP in the network at steady state among the components over this experimental range and an extrapolated range of stimulation frequencies (up to 10 Hz). The control analysis showed that the contractile actomyosin ATPase has dominant kinetic control of ATP flux in forearm flexor muscle over the 0- to 1.6-Hz range of contraction frequencies that resulted in steady states, as determined by (31)P-NMR. However, flux control begins to shift toward mitochondria at >1 Hz. This inversion of flux control from ATP demand to ATP supply control hierarchy progressed as the contraction frequency increased past 2 Hz and was nearly complete at 10 Hz. The functional significance of this result is that, at steady state, ATP free energy consumption cannot outstrip the ATP free energy supply. Therefore, this reduced, three-component muscle ATPase system is inherently homeostatic.

Cellular energetics analysis by a mathematical model of energy balance: estimation of parameters in human skeletal muscle.

Cellular energy balance requires that the physiological demands by ATP-utilizing functions be matched by ATP synthesis to sustain muscle activity. We devised a new method of analysis of these processes in data from single individuals. Our approach is based on the logic of current information on the major mechanisms involved in this energy balance and can quantify not directly measurable parameters that govern those mechanisms. We use a mathematical model that simulates by ordinary, nonlinear differential equations three components of cellular bioenergetics (cellular ATP flux, mitochondrial oxidative phosphorylation, and creatine kinase kinetics). We incorporate data under resting conditions, during the transition toward a steady state of stimulation and during the transition during recovery back to the original resting state. Making use of prior information about the kinetic parameters, we fitted the model to previously published dynamic phosphocreatine (PCr) and inorganic phosphate (P(i)) data obtained in normal subjects with an activity-recovery protocol using (31)P nuclear magnetic resonance spectroscopy. The experiment consisted of a baseline phase, an ischemic phase (during which muscle stimulation and PCr utilization occurred), and an aerobic recovery phase. The model described satisfactorily the kinetics of the changes in PCr and P(i) and allowed estimation of the maximal velocity of oxidative phosphorylation and of the net ATP flux in individuals both at rest and during stimulation. This work lays the foundation for a quantitative, model-based approach to the study of in vivo muscle energy balance in intact muscle systems, including human muscle.

Energy balance in muscle activity: simulations of ATPase coupled to oxidative phosphorylation and to creatine kinase.

Energy balance refers to the dynamic homeostasis of ATP and related forms of chemical potential within cells. This regulation is accomplished mainly by oxidative metabolism in most mammals. This homeostasis matches dynamically the energy demands of cellular ATPases (net decrease in chemical potential energy) with the energy supply by mitochondrial oxidative phosphorylation (net increase in chemical potential energy). Muscle cells are distinguished from most other cell types in their ability to attain energy balance with more than a 10-fold range of ATPase demand. Creatine kinase maintains a near to equilibrium flux: PCr + ADP<-->ATP + Cr. One important function of creatine kinase is to buffer ATP and ADP concentrations. A system of differential equations describe the coupled operation of cellular ATPase, creatine kinase and oxidative phosphorylation. These equations used experimentally measured concentrations of relevant metabolites and enzyme activities to simulate energy balance in muscle cells. The principle of energy balance is adequately illustrated by simulations with only a three component system.

Glycolysis is independent of oxygenation state in stimulated human skeletal muscle in vivo.

1. We tested the hypothesis that the cytoplasmic control mechanism for glycolysis is affected by the presence of oxygen during exercise. We used a comparison of maximal twitch stimulation under ischaemic and intact circulation in human wrist flexor and ankle dorsiflexor muscles. 31P magnetic resonance spectroscopy followed the phosphocreatine (PCr), Pi and pH dynamics at 6-9 s intervals. Glycolytic PCr synthesis was determined during stimulation from pH and tissue buffer capacity, as well as the oxidative phosphorylation rate. 2. Ischaemic vs. aerobic stimulation resulted in similar glycolytic fluxes in the two muscles. The onset of glycolysis occured after fifty to seventy stimulations and the extent of glycolytic PCr synthesis was directly proportional to the number of stimulations thereafter. 3. Two-fold differences in the putative feedback regulators of glycolysis, [Pi] and [ADP], were found between aerobic and ischaemic stimulation. The similar glycolytic fluxes in the face of these differences in metabolite levels eliminates feedback as a control mechanism in glycolysis. 4. These results demonstrate that glycolytic flux is independent of oxygenation state and metabolic feedback, but proportional to muscle activation. These results show a key role for muscle stimulation in the activation and maintenance of glycolysis. Further, this glycolytic control mechanism is independent of the feedback control mechanism that governs oxidative phosphorylation.

Effect of viscosity on mechanics of single, skinned fibers from rabbit psoas muscle.

Muscle contraction is highly dynamic and thus may be influenced by viscosity of the medium surrounding the myofilaments. Single, skinned fibers from rabbit psoas muscle were used to test this hypothesis. Viscosity within the myofilament lattice was increased by adding to solutions low molecular weight sugars (disaccharides sucrose or maltose or monosaccharides glucose or fructose). At maximal Ca2+ activation, isometric force (Fi) was inhibited at the highest solute concentrations studied, but this inhibition was not directly related to viscosity. Solutes readily permeated the filament lattice, as fiber diameter was unaffected by added solutes (except for an increased diameter with Fi < 30% of control). In contrast, there was a linear dependence upon 1/viscosity for both unloaded shortening velocity and also the kinetics of isometric tension redevelopment; these effects were unrelated to either variation in solution osmolarity or inhibition of force. All effects of added solute were reversible. Inhibition of both isometric as well as isotonic kinetics demonstrates that viscous resistance to filament sliding was not the predominant factor affected by viscosity. This was corroborated by measurements in relaxed fibers, which showed no significant change in the strain-rate dependence of elastic modulus when viscosity was increased more than twofold. Our results implicate cross-bridge diffusion as a significant limiting factor in cross-bridge kinetics and, more generally, demonstrate that viscosity is a useful probe of actomyosin dynamics.

Non-invasive quantitative 31P MRS assay of mitochondrial function in skeletal muscle in situ.

A method for non-invasive, quantitative 31P NMR spectroscopic investigation of mitochondrial function in human skeletal muscle in situ is described. High time resolution 31P NMR measurements of phosphocreatine, inorganic phosphate and ATP resonances were conducted on human forearm flexor muscle during involuntary twitch contraction at eight different frequencies. Mitochondrial and glyco(geno)lytic ATP synthesis fluxes, and the cytosolic free energy of ATP hydrolysis (delta GP), were calculated at incremental steady-states of energy balance. The covariation of mitochondrial ATP synthesis flux, JPMOP, and delta GP was quasi-linear over the physiological range of free energy values. Curve-fit analysis of the covariation yielded a maximal sustainable JPMOP of 0.24 +/- 0.06 mmol ATP l-1.s-1 and a midpoint potential, (delta GP)0.5, of 58.1 +/- 1.2 kJ/mole in the muscle cells.

Phosphorus metabolite distribution in skeletal muscle: quantitative bioenergetics using creatine analogs.

The functional coupling of contractile activity to metabolic processes in skeletal, cardiac and smooth muscle has been extensively examined in the intact cell through the advent of 31P-NMR spectroscopy. The near-equilibrium formulation for creatine kinase (CK) has been used for the calculation of ADPfree and ATP chemical potential in many of these studies. However, control of the bulk cytoplasmic PCr/Cr ratio by the ATP/ADP ratio through CK implies that the ATP/ADP ratio is the same in all loci within the cell. Alternatively the cytoplasmic fraction of ATP and ADP must be so large that other 'compartments' do not influence the physicochemical properties of the bulk cytoplasm. By feeding creatine analogs to rodents, it is possible to test whether these synthetic analogs and the endogenous substrates obey simple rules of enzyme kinetics and equilibration. Two important concepts can be tested: (1) Are phosphorus metabolises fully visible to in vivo 31P-NMR measurements? (2) Does CK equilibrate with its substrates in the cytosol? It will be shown that in spite of localized enzymatic activity and microcompartments in the cell (such as mitochondria), creatine kinase equilibrates with its substrates in fast and slow skeletal muscles at rest. Therefore, the physicochemical properties of the cytosol are best described as freely mixing with respect to the bioenergetically important metabolites (PCr, ATP and phospho-analog) and fully quantifiable by 31P-NMR spectroscopy. It follows that only a narrow range of intercellular heterogeneity with respect to chemical potential is acceptable if bioenergetic processes (e.g. CK fluxes or regulation of oxidative phosphorylation) are to be meaningfully interpreted in a rigorous biochemical framework.

Activation of glycolysis in human muscle in vivo.

We tested the cytoplasmic control mechanisms for glycolytic ATP synthesis in human wrist flexor muscles. The forearm was made ischemic and activated by maximal twitch stimulation of the median and ulnar nerves in 10 subjects. Kinetic changes in phosphocreatine, Pi, ADP, ATP, sugar phosphates, and pH were measured by 31P magnetic resonance spectroscopy at 7.1-s intervals. Proton production was determined from pH and tissue buffer capacity during stimulation. Glycolysis was activated between 30 and 50 stimulations, and the rate did not significantly change through the stimulation period. The independence of glycolytic rate on [Pi], [ADP], or [AMP] indicates that feedback regulation by these metabolites could not account for this activation of glycolysis. However, glycolytic H+ and ATP production increased sixfold from 0.5 to 3 Hz, indicating that glycolytic rate reflected muscle activation frequency. This dependence of glycolytic rate on muscle stimulation frequency and independence on metabolite levels is consistent with control of glycolysis by Ca2+.

Skeletal muscle perfusion measurements using adiabatic inversion of arterial water.

Quantitative NMR measurements of perfusion using magnetic labeling of arterial water have been demonstrated previously in several different highly perfused organs. The success of these previous experiments suggested that arterial labeling may be of use in measuring perfusion in skeletal muscle, where resting perfusion is very low and where increased perfusion after exercise is transient. In the experiments described in this paper, adiabatic inversion of arterial water has been used to make single-voxel measurements of perfusion in the lower hind limb of rats. At rest, the NMR results were quantified to yield a perfusion rate of about 13.8 ml/100g/min. After perturbation due to ischemic exercise, large relative changes in the NMR signal were observed. The peak change of about 2.5% of the NMR signal occurred shortly after perturbation and was followed by a return to resting levels over a period of about 4 min.

Multiple equilibria of cations with metabolites in muscle bioenergetics.

The multiple ionic forms of metabolites were evaluated at 37 degrees C for four reactions important in muscle contraction and recovery: 1) ATPase, 2) creatine kinase, 3) the Lohmann reaction, and 4) the Lohmann reaction reversed by coupling to glycogenolysis and glycolysis. Solution of the system of equations defining the multiple equilibria of the proton and cation complexes gives the concentration of each ionic form and a value for the proton stoichiometry for each reaction. The proton stoichiometric coefficients are unique for each reaction and are a function of pH because of differential binding of Mg2+ and K+ to adenine nucleotides, phosphocreatine, and Pi and because of different acidic dissociation constants for the metabolites. These results show the need to consider the binding of K+ in addition to the previously documented effects of Mg2+ in the cytoplasmic milieu. Commercially available software was used to show that related problems can be calculated readily on personal computers in applications similar to those described here.

Recovery after contraction of white muscle fibres from the dogfish Scyliorhinus canicula.

Recovery after contraction of white muscle fibres of dogfish was investigated using 31P-NMR and measurements of heat production. The muscle fibres were stimulated to perform either a single isometric tetanus or a series of brief isometric tetani; the NMR measurements showed that approximately half of the phosphocreatine (PCr) was used. The period of activity was followed by a recovery period without stimulation. Both NMR and heat measurements agreed in showing that recovery was very slow, requiring at least 60 min for PCr resynthesis and for the production of recovery heat. The NMR results showed that changes in intracellular pH and in the concentrations of PCr and intracellular phosphate (Pi) had very similar time courses. Intracellular pH moved in the alkaline direction during the period of activity and then returned monotonically during recovery. The non-phosphate buffer power was 13.0 +/- 3.1 mmol l-1 intracellular water per pH unit (N = 4, mean +/- S.E.M.). The results are consistent with the view that oxidative processes resynthesize PCr during recovery, which is slow because of the low mitochondrial content of these muscle fibres.

A model of the inversion process in an arterial inversion experiment.

A model of the behavior of spins moving through spatially varying gradient and B1 fields is presented. The model simulates the adiabatic behavior of flowing arterial water during a two-coil arterial inversion experiment. Predictions of the degree of inversion generated by the model are compared with flow phantom results for a wide range of gradient magnitudes, nominal B1 magnitudes, and flow velocities. The high level of agreement between the model and the flow phantom results indicates that the model can be used to help select efficient pulse sequence parameters when setting up an in vivo arterial inversion experiment. In addition, the model provides valuable insights into the adiabatic behavior of arterial spins. These insights could be useful in selecting an efficient surface coil geometry which achieves maximum inversion with a minimum B1 magnitude.

The signal transduction function for oxidative phosphorylation is at least second order in ADP.

To maintain ATP constant in the cell, mitochondria must sense cellular ATP utilization and transduce this demand to F0-F1-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 31P 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.

Activity-dependent induction of slow myosin gene expression in isolated fast-twitch mouse muscle.

We demonstrate that direct electrical stimulation of isolated fast-twitch muscle in an organ culture system can induce expression of the slow myosin heavy chain (beta-MHC) gene, indicative of a phenotype transformation. Pairs of extensor digitorum longus (EDL) muscles were isolated from adult mice, incubated at resting length in separate chambers, and superfused with the same recirculated media One muscle was subjected to twitch stimulation (5-s trains of 5-Hz pulses at supramaximal voltage every minute), and force was recorded to assess function. The contralateral muscle was incubated without stimulation, to control for effects of the experimental preparation. Both muscle were rapidly frozen for RNA purification and oligo(dT)-primed reverse transcription; serial studies were carried out to 36 h. Polymerase chain reaction was performed utilizing primers specific for cytoplasmic beta-actin (beta-actin), a constitutive marker, and beta-MHC, a gene that is either inactive or expressed at very low levels in control EDL. After 30 h of stimulation, beta-MHC was consistently detected at a level severalfold higher in stimulated EDL than in incubated control EDL when band intensities were normalized to those of beta-actin. These results show that signals or fiber-specific transformations reside within the muscle and that this shift begins rapidly after induction of continuous stimulation.