Impaired oxidative phosphorylation in hepatic mitochondria in growth-retarded rats

Intrauterine growth retardation (IUGR) has been linked to the development of type 2 diabetes in adulthood. We have developed an IUGR model in the rat whereby the animals develop diabetes between 3 and 6 mo of age that is associated with insulin resistance. Alterations in hepatic glucose metabolism are known to contribute to the hyperglycemia of diabetes; however, the mechanisms underlying this phenomenon have not been fully explained. To address this issue, intact liver mitochondria were isolated from IUGR and control offspring at different ages to examine the nature and time course of possible defects in oxidative metabolism. Phosphoenolpyruvate carboxykinase (PEPCK) expression was also measured in livers of IUGR and control offspring. Rates of ADP-stimulated (state 3) oxygen consumption were increased for succinate in the fetus and for alpha-ketoglutarate and glutamate at day 1, reflecting possible compensatory metabolic adaptations to acute hypoxia and acidosis in IUGR rats. By day 14, oxidation of glutamate and alpha-ketoglutarate had returned to normal, and by day 28, oxidation rates of pyruvate, glutamate, succinate, and alpha-ketoglutarate were significantly lower than those of controls. Rotenone-sensitive NADH-O2 oxidoreductase activity was similar in control and IUGR mitochondria at all ages, showing that the defect responsible for decreased pyruvate, glutamate, and alpha-ketoglutarate oxidation in IUGR liver precedes the electron transport chain and involves pyruvate and alpha-ketoglutarate dehydrogenases. Increased levels of manganese superoxide dismutase suggest that an antioxidant response has been mounted, and hydroxynonenal (HNE) modification of pyruvate dehydrogenase E2-(catalytic) and E3-binding protein subunits suggests that HNE-induced inactivation of this key enzyme may play a role in the mechanism of injury. The level of PEPCK mRNA was increased 250% in day 28 IUGR liver, indicating altered gene expression of the gluconeogenic enzyme that precedes overt hyperglycemia. These results indicate that uteroplacental insufficiency impairs mitochondrial oxidative phosphorylation in the liver and that this derangement predisposes the IUGR rat to increased hepatic glucose production by suppressing pyruvate oxidation and increasing gluconeogenesis.     

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.     

Control of oxidative phosphorylation in skeletal muscle

The classical concept of ATP-demand control of energy metabolism in skeletal muscle has to be modified on the basis of studies showing the influence of additional controlling parameters (reducing equivalent supply, oxygen availability, proton leak, diffusion restrictions and the creatine kinase system) and on the basis of applications of metabolic control analysis showing very clearly multistep control. This concept of multistep control allows to quantify the individual influence of any parameter on mitochondrial oxidative phosphorylation and is extremely helpful to analyze the metabolic consequences of enzyme deficiencies in skeletal muscle occurring in mitochondrial myopathies.     

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.     

Impaired skeletal muscle perfusion in obese Zucker rats

Skeletal muscle arterioles from obese Zucker rats (OZR) exhibit oxidant stress-based alterations in reactivity, enhanced alpha-adrenergic constriction, and reduced distensibility vs. microvessels of lean Zucker rats (LZR). The present study determined the impact of these alterations for perfusion and performance of in situ skeletal muscle during periods of elevated metabolic demand. During bouts of isometric tetanic contractions, fatigue of in situ gastrocnemius muscle of OZR was increased vs. LZR; this was associated with impaired active hyperemia. In OZR, vasoactive responses of skeletal muscle arterioles from the contralateral gracilis muscle were impaired, due in part to elevated oxidant tone; reactivity was improved after treatment with polyethylene glycol-superoxide dismutase (PEGSOD). Arterioles of OZR also exhibited increased alpha-adrenergic sensitivity, which was abolished by treatment with phentolamine (10-5 M). Intravenous infusion of phentolamine (10 mg/kg) or PEG-SOD (2,000 U/kg) in OZR altered neither fatigue rates nor active hyperemia from untreated levels; however, combined infusion improved performance and hyperemia, although not to levels in LZR. Microvessel density in the contralateral gastrocnemius muscle, determined via histological analyses, was reduced by approximately 25% in OZR vs. LZR, while individual arterioles from the contralateral gracilis muscle demonstrated reduced distensibility. These data suggest that altered arteriolar reactivity contributes to reduced muscle performance and active hyperemia in OZR. Further, despite pharmacological improvements in arteriolar reactivity, reduced skeletal muscle microvessel density and arteriolar distensibility also contribute substantially to reduced active hyperemia and potentially to impaired muscle performance.     

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 capillary hemodynamics in skeletal muscle of rats in chronic heart failure.

Skeletal muscle blood flow is reduced and O(2) extraction is increased at rest in chronic heart failure (CHF). Knowledge of red blood cell (RBC) flow distribution within the capillary network is necessary for modeling O(2) delivery and exchange in this disease. Intravital microscopy techniques were used to study the in vivo spinotrapezius muscle microcirculation in rats with CHF 7 wk after myocardial infarction and in sham-operated controls (sham). A decrease in mean muscle fiber width from 51.3 +/- 1.9 microm in sham to 42.6 +/- 1.4 microm in CHF rats (P < 0.01) resulted in an increased lineal density of capillaries in CHF rats (P < 0.05). CHF reduced (P < 0.05) the percentage of capillaries supporting continuous RBC flow from 87 +/- 5 to 66 +/- 5%, such that the lineal density of capillaries supporting continuous RBC flow remained unchanged. The percentage of capillaries supporting intermittent RBC flow was increased in CHF rats (8 and 27% in sham and CHF, respectively, P < 0.01); however, these capillaries contributed only 2.3 and 3.3% of the total RBC flux in sham and CHF rats, respectively. In continuously RBC-perfused capillaries, RBC velocity (252 +/- 20 and 144 +/- 9 microm/s in sham and CHF, respectively, P < 0.001) and flux (21.4 +/- 2.4 and 9.4 +/- 1.1 cells/s in sham and CHF, respectively, P < 0.01) were markedly reduced in CHF compared with sham rats. Capillary "tube" hematocrit remained unchanged (0.22 +/- 0.02 and 0.19 +/- 0.02 in sham and CHF, respectively, P > 0.05). We conclude that CHF causes spinotrapezius fiber atrophy and reduces the number of capillaries supporting continuous RBC flow per fiber. Within these capillaries supporting continuous RBC flow, RBC velocity and flux are reduced 45-55%. This decreases the potential for O(2) delivery but enhances fractional O(2) extraction by elevating RBC capillary residence time. The unchanged capillary tube hematocrit suggests that any alterations in muscle O(2) diffusing properties in CHF are mediated distal to the RBC.     

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.     

Impaired NO-dependent dilation of skeletal muscle arterioles in hypertensive diabetic obese Zucker rats

This study determined alterations to nitric oxide (NO)-dependent dilation of skeletal muscle arterioles from obese (OZR) versus lean Zucker rats (LZR). In situ cremaster muscle arterioles from both groups were viewed via television microscopy, and vessel dilation was measured with a video micrometer. Arteriolar dilation to acetylcholine and sodium nitroprusside was reduced in OZR versus LZR, although dilation to aprikalim was unaltered. NO-dependent flow-induced arteriolar dilation (via parallel microvessel occlusion) was attenuated in OZR, impairing arteriolar ability to regulate wall shear rate. Vascular superoxide levels, as assessed by dihydroethidine fluorescence, were elevated in OZR versus LZR. Treatment of cremaster muscles of OZR with the superoxide scavengers polyethylene glycol-superoxide dismutase and catalase improved arteriolar dilation to acetylcholine and sodium nitroprusside and restored flow-induced dilation and microvascular ability to regulate wall shear rate. These results suggest that NO-dependent dilation of skeletal muscle microvessels in OZR is impaired due to increased levels of superoxide. Taken together, these data suggest that the development of diabetes and hypertension in OZR may be associated with an impaired skeletal muscle perfusion via an elevated vascular oxidant stress.     

Impaired skeletal muscle microcirculation in systemic sclerosis

Introduction

Muscle symptoms in systemic sclerosis (SSc) may originate from altered skeletal muscle microcirculation, which can be investigated by means of blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI).

Methods

After ethics committee approval and written consent, 11 consecutive SSc patients (5 men, mean age 52.6 years, mean SSc disease duration 5.4 years) and 12 healthy volunteers (4 men, mean age 45.1 years) were included. Subjects with peripheral arterial occlusive disease were excluded. BOLD MRI was performed on calf muscles during cuff-induced ischemia and reactive hyperemia, using a 3-T whole-body scanner (Verio, Siemens, Erlangen, Germany) and fat-suppressed single-short multi-echo echo planar imaging (EPI) with four different effective echo times. Muscle BOLD signal time courses were obtained for gastrocnemius and soleus muscles: minimal hemoglobin oxygen saturation (T2*_min) and maximal T2* values (T2*_max), time to T2* peak (TTP), and slopes of oxygen normalization after T2* peaking.

Results

The vast majority of SSc patients lacked skeletal muscle atrophy, weakness or serum creatine kinase elevation. Nevertheless, more intense oxygen desaturation during ischemia was observed in calf muscles of SSc patients (mean T2*_min -15.0%), compared with controls (-9.1%, P = 0.02). SSc patients also had impaired oxygenation during hyperemia (median T2*_max 9.2% vs. 20.1%, respectively, P = 0.007). The slope of muscle oxygen normalization was significantly less steep and prolonged (TTP) in SSc patients (P<0.001 for both). Similar differences were found at a separate analysis of gastrocnemius and soleus muscles, with most pronounced impairment in the gastrocnemius.

Conclusions

BOLD MRI demonstrates a significant impairment of skeletal muscle microcirculation in SSc.     

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.     

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.     

Role of mitochondria in exercise-induced oxidative stress in skeletal muscle from hyperthyroid rats

Previous study showed that exercise induces higher oxidative damage and respiratory capacity reduction in hyperthyroid than in euthyroid skeletal muscle. Because impaired cell function can result from mitochondrial dysfunction, we evaluated the changes induced by exercise in oxygen consumption of skeletal muscle mitochondria from euthyroid and hyperthyroid rats. The mitochondrial function was related with indices of oxidative damage and nitric oxide production, scavenger levels and mitochondrial ROS production rates. Our results show that exercise increased state 4 and decreased state 3 respiration, and the highest changes happened in hyperthyroid preparations. This was consistent with the observation that oxidative damage and NO(*) derivative content were increased by T(3) administration and exercise, reaching the highest levels in hyperthyroid exercised rats. Our results also indicate that the high mitochondrial oxidative damage induced by T(3) and exercise is due to enhanced ROS production, which is dependent on increases in mitochondrial content and reduction degree, respectively, of autoxidizable electron carriers.     

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)