A model of oxidative phosphorylation in mammalian skeletal muscle

A dynamic computer model of oxidative phosphorylation in oxidative mammalian skeletal muscle was developed. The previously published model of oxidative phosphorylation in isolated skeletal muscle mitochondria was extended by incorporation of the creatine kinase system (creatine kinase plus phosphocreatine/creatine pair), cytosolic proton production/consumption system (proton production/consumption by the creatine kinase-catalysed reaction, efflux/influx of protons), physiological size of the adenine nucleotide pool and some additional minor changes. Theoretical studies performed by means of the extended model demonstrated that the CK system, which allows for large changes in P(i) in relation to isolated mitochondria system, has no significant influence on the kinetic properties of oxidative phosphorylation, as inorganic phosphate only slightly modifies the relationship between the respiration rate and [ADP]. Computer simulations also suggested that the second-order dependence of oxidative phosphorylation on [ADP] proposed in the literature refers only to the ATP synthesis flux, but not to the oxygen consumption flux (the difference between these two fluxes being due to the proton leak). Next, time courses of changes in fluxes and metabolite concentrations during transition between different steady-states were simulated. The model suggests, in accordance with previous theoretical predictions, that activation of oxidative phosphorylation by an increase in [ADP] can (roughly) explain the behaviour of the system only at low work intensities, while at higher work intensities parallel activation of different steps of oxidative phosphorylation is involved.     

Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria

Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.     

Impaired oxidative phosphorylation in skeletal muscle of intrauterine growth-retarded rats

Intrauterine growth retardation (IUGR) has been linked to the development of type 2 diabetes in later life. We have developed a model of uteroplacental insufficiency, a common cause of intrauterine growth retardation, in the rat. Early in life, the animals are insulin resistant and by 6 mo of age they develop diabetes. Glycogen content and insulin-stimulated 2-deoxyglucose uptake were significantly decreased in muscle from IUGR rats. IUGR muscle mitochondria exhibited significantly decreased rates of state 3 oxygen consumption with pyruvate, glutamate, alpha-ketoglutarate, and succinate. Decreased pyruvate oxidation in IUGR mitochondria was associated with decreased ATP production, decreased pyruvate dehydrogenase activity, and increased expression of pyruvate dehydrogenase kinase 4. Such a defect in IUGR mitochondria leads to a chronic reduction in the supply of ATP available from oxidative phosphorylation. Impaired ATP synthesis in muscle compromises energy-dependent GLUT4 recruitment to the cell surface, glucose transport, and glycogen synthesis, which contribute to insulin resistance and hyperglycemia of type 2 diabetes.     

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca²⁺ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Capacity of oxidative phosphorylation in human skeletal muscle: New perspectives of mitochondrial physiology

Maximal ADP-stimulated mitochondrial respiration depends on convergent electron flow through Complexes I + II to the Q-junction of the electron transport system (ETS). In most studies of respiratory control in mitochondrial preparations, however, respiration is limited artificially by supplying substrates for electron input through either Complex I or II. High-resolution respirometry with minimal amounts of tissue biopsy (1–3 mg wet weight of permeabilized muscle fibres per assay) provides a routine approach for multiple substrate-uncoupler-inhibitor titrations. Under physiological conditions, maximal respiratory capacity is obtained with glutamate + malate + succinate, reconstituting the operation of the tricarboxylic acid cycle and preventing depletion of key metabolites from the mitochondrial matrix. In human skeletal muscle, conventional assays with pyruvate + malate or glutamate + malate yield submaximal oxygen fluxes at 0.50–0.75 of capacity of oxidative phosphorylation (OXPHOS). Best estimates of muscular OXPHOS capacity at 37 °C (pmol O₂ s⁻¹ mg⁻¹ wet weight) with isolated mitochondria or permeabilized fibres, suggest a range of 100–150 and up to 180 in healthy humans with normal body mass index and top endurance athletes, but reduction to 60–120 in overweight healthy adults with predominantly sedentary life style. The apparent ETS excess capacity (uncoupled respiration) over ADP-stimulated OXPHOS capacity is high in skeletal muscle of active and sedentary humans, but absent in mouse skeletal muscle. Such differences of mitochondrial quality in skeletal muscle are unexpected and cannot be explained at present. A comparative database of mitochondrial physiology may provide the key for understanding the functional implications of mitochondrial diversity from mouse to man, and evaluation of altered mitochondrial respiratory control patterns in health and disease.     

Changes of the oxidative phosphorylation in mitchondria of rat skeletal muscle following strenous exercise.

The rate of oxygen consumption of mitochondria from rat muscles at pH 7.4 is elevated by 1-lactate. The respiratory control ratio and the ADP/O-ratio are decreased under these conditions. Acidification to pH 6.5 in the absence of 1-lactate does not change the interpreted mitochondrial functions. The experimental data are discussed as a partial uncoupling effect of 1-lactate on the oxidative phosphorylation. Similar changes in those mitochondrial functions are found after short-time intensive swimming exercise of rats. These variations might be a reason for the sometimes described reduced aerobic performance after intensive work.     

Alteration of mitochondrial oxidative phosphorylation in aged skeletal muscle involves modification of adenine nucleotide translocator

The process of skeletal muscle aging is characterized by a progressive loss of muscle mass and functionality. The underlying mechanisms are highly complex and remain unclear. This study was designed to further investigate the consequences of aging on mitochondrial oxidative phosphorylation in rat gastrocnemius muscle, by comparing young (6 months) and aged (21 months) rats. Maximal oxidative phosphorylation capacity was clearly reduced in older rats, while mitochondrial efficiency was unaffected. Inner membrane properties were unaffected in aged rats since proton leak kinetics were identical to young rats. Application of top-down control analysis revealed a dysfunction of the phosphorylation module in older rats, responsible for a dysregulation of oxidative phosphorylation under low activities close to in vivo ATP turnover. This dysregulation is responsible for an impaired mitochondrial response toward changes in cellular ATP demand, leading to a decreased membrane potential which may in turn affect ROS production and ion homeostasis. Based on our data, we propose that modification of ANT properties with aging could partly explain these mitochondrial dysfunctions.     

Age-associated deficit of mitochondrial oxidative phosphorylation in skeletal muscle: Role of carnitine and lipoic acid

Mitochondrial damage has implicated a major contributor for ageing process. In the present study, we measured mitochondrial membrane swelling, mitochondrial respiration (state 3 and 4) by using oxygen electrode in skeletal muscle of young (3–4 months old) and aged rats (above 24 months old) with supplementation of l-carnitine and dl-α-lipoic acid. Our results shows that the mitochondrial membrane swelling and state 4 respiration were increased more in skeletal muscle mitochondria of aged rats than in young control rats, whereas the state 3 respiration, respiratory control ratio (RCR) and ADP:O ratio decreased more in aged rats than in young rats. After supplementation of carnitine and lipoic acid to aged rats for 30 days, the state 3 respiration and RCR were increased, whereas the state 4 and mitochondrial membrane swelling were decreased to near normal rats. From our results, we conclude that combined supplementation of carnitine and lipoic acids to aged rats increases the skeletal muscle mitochondrial respiration, thereby increasing the level of ATP. (Mol Cell Biochem xxx: 83–89, 2005)     

Control of mitochondrial oxidative phosphorylation

The objective of this investigation is to analyze the two following problems of the regulation of mitochondrial oxidative phosphorylation: what is the extramitochondrial parameter that controls ATP production according to the cytoplasmic demands and how the control is distributed between various mitochondrial enzymes. On the basis of the data of Groen et al. (1982) it is shown that as the respiration rates ranged over 30-50% of the maximum (i.e. within the physiological region) the contribution of the adenine nucleotide translocator to the control of the ATP flux is no less than 90%, referring to the total contribution of all mitochondrial enzymes as 100%. Founding on the key role of the adenine nucleotide translocator it has been concluded that besides the extramitochondrial [ATP]/[ADP] ratio the absolute ADP concentration is another extramitochondrial signal controlling significantly the rate of oxidative phosphorylation.     

Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis

In skeletal muscle, two mitochondrial populations are present which, on the basis of their localisation, are termed intermyofibrillar and subsarcolemmal mitochondria (IMF and SS, respectively). These two populations have different biochemical characteristics and show different responses to physiological stimuli. In this paper, we characterise the oxidative phosphorylation of SS and IMF using 'top-down' elasticity analysis. We excluded the possibility that their different characteristics can be attributed to a different degree of breakage of the two types of mitochondria due to the different isolation procedures used in their preparation. The higher respiration rate and higher respiratory control ratio shown by IMF compared with those shown by SS are principally due to the higher activities of the reactions involved in substrate oxidation as confirmed by the measurement of cytochrome oxidase activity. There is no difference in the leak of protons across the inner mitochondrial membrane between IMF and SS; a faster rate of ATP synthesis and turnover is driven by the lower membrane potential in SS compared with in IMF.     

Increased intramyocellular lipids but unaltered in vivo mitochondrial oxidative phosphorylation in skeletal muscle of adipose triglyceride lipase-deficient mice

Adipose triglyceride lipase (ATGL) is a lipolytic enzyme that is highly specific for triglyceride hydrolysis. The ATGL-knockout mouse (ATGL(-/-)) accumulates lipid droplets in various tissues, including skeletal muscle, and has poor maximal running velocity and endurance capacity. In this study, we tested whether abnormal lipid accumulation in skeletal muscle impairs mitochondrial oxidative phosphorylation, and hence, explains the poor muscle performance of ATGL(-/-) mice. In vivo 1H magnetic resonance spectroscopy of the tibialis anterior of ATGL(-/-) mice revealed that its intramyocellular lipid pool is approximately sixfold higher than in WT controls (P = 0.0007). In skeletal muscle of ATGL(-/-) mice, glycogen content was decreased by 30% (P < 0.05). In vivo 31P magnetic resonance spectra of resting muscles showed that WT and ATGL(-/-) mice have a similar energy status: [PCr], [P(i)], PCr/ATP ratio, PCr/P(i) ratio, and intracellular pH. Electrostimulated muscles from WT and ATGL(-/-) mice showed the same PCr depletion and pH reduction. Moreover, the monoexponential fitting of the PCr recovery curve yielded similar PCr recovery times (τPCr; 54.1 ± 6.1 s for the ATGL(-/-) and 58.1 ± 5.8 s for the WT), which means that overall muscular mitochondrial oxidative capacity was comparable between the genotypes. Despite similar in vivo mitochondrial oxidative capacities, the electrostimulated muscles from ATGL(-/-) mice displayed significantly lower force production and increased muscle relaxation time than the WT. These findings suggest that mechanisms other than mitochondrial dysfunction cause the impaired muscle performance of ATGL(-/-) mice.     

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ～95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.     

Application of modular kinetic analysis to mitochondrial oxidative phosphorylation in skeletal muscle of birds exposed to acute heat stress

We previously showed that heat stress stimulates reactive oxygen species (ROS) production in skeletal muscle mitochondria of birds, probably via an elevation in mitochondrial membrane potential (ΔΨ). To clarify the mechanism underlying the elevation of ΔΨ, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of heat-stressed birds (34 °C for 12 h). In the birds exposed to heat stress, ‘substrate oxidation’ (a ΔΨ-producer) was increased compared to control (24 °C) birds, although there was little difference in ‘proton leak’ (a ΔΨ-consumer), suggesting that an elevation in the ΔΨ at state 4 may be due to enhanced substrate oxidation. It thus appears that enhanced substrate oxidation plays a crucial role in the overproduction of ROS for heat-stressed birds, probably via elevated ΔΨ.