Search

Most of the cell's energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in any given organ or tissue. Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Depending on metabolic requirements, skeletal muscle mitochondria integrate O 2 consumption and ATP production with lipid, glucose, or amino acid metabolism. Free fatty acids (FFAs) are the main source of energy during rest and mild-intensity exercise. We present a detailed protocol for measuring FFA-β-oxidation coupled with O 2 respiration by a Clark-type electrode in isolated mitochondria from mouse soleus oxidative muscle. We optimized the procedure, including buffer composition, protease treatment, and quantifiable parameters (P/O, Phosphate/Oxygen Ratio; OCR, Oxygen Consumption Rate; RCR,Respiration Control Rate; OSR, Oligomycin Sensitive Respiration). For complete details on the use and execution of this protocol, please refer to Sanchez-Gonzalez et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Purpose: This study aimed to evaluate the influence of lifelong regular physical activity on skeletal muscle capillarization in women., Methods: Postmenopausal women, 61±4 yr old, were divided according to self-reported physical activity level over the past 20 yrs: sedentary (SED; n = 14), moderately active (MOD; n = 12), and very active (VERY; n = 15). Leg blood flow (LBF) was determined by ultrasound Doppler, and blood samples were drawn from the femoral artery and vein for calculation of leg oxygen uptake (LVO2) at rest and during one-legged knee extensor exercise. A skeletal muscle biopsy was obtained from the vastus lateralis and analyzed for capillarization and vascular endothelial growth factor (VEGF) and mitochondrial OXPHOS proteins. Platelets were isolated from venous blood and analyzed for VEGF content and effect on endothelial cell proliferation., Results: The exercise-induced rise in LBF and LVO2 was faster (P = 0.008) in VERY compared with SED and MOD. Steady-state LBF and LVO2 were lower (P < 0.04) in MOD and VERY compared with SED. Capillary-fiber ratio and capillary density were greater (P < 0.03) in VERY (1.65 ± 0.48 and 409.3 ± 57.5) compared with MOD (1.30 ± 0.19 and 365.0 ± 40.2) and SED (1.30 ± 0.30 and 356.2 ± 66.3). Skeletal muscle VEGF and OXPHOS complexes I, II, and V were ~1.6-fold and ~1.25-fold (P < 0.01) higher, respectively, in VERY compared with SED. Platelets from all groups induced an approximately nine-fold (P < 0.001) increase in endothelial cell proliferation., Conclusion: A very active lifestyle is associated with superior skeletal muscle exercise hemodynamics and greater potential for oxygen extraction concurrent with a higher skeletal muscle capillarization and mitochondrial capacity., (Copyright © 2021 by the American College of Sports Medicine.)

Aims/hypothesis: Subcellular localisation is an important factor in the known impact of bioactive lipids, such as diacylglycerol and sphingolipids, on insulin sensitivity in skeletal muscle; yet, the role of localised intramuscular triacylglycerol (IMTG) is yet to be described. Excess accumulation of IMTG in skeletal muscle is associated with insulin resistance, and we hypothesised that differences in subcellular localisation and composition of IMTG would relate to metabolic health status in humans., Methods: We evaluated subcellular localisation of IMTG in lean participants, endurance-trained athletes, individuals with obesity and individuals with type 2 diabetes using LC-MS/MS of fractionated muscle biopsies and insulin clamps., Results: Insulin sensitivity was significantly different between each group (athletes>lean>obese>type 2 diabetes; p < 0.001). Sarcolemmal IMTG was significantly greater in individuals with obesity and type 2 diabetes compared with lean control participants and athletes, but individuals with type 2 diabetes were the only group with significantly increased saturated IMTG. Sarcolemmal IMTG was inversely related to insulin sensitivity. Nuclear IMTG was significantly greater in individuals with type 2 diabetes compared with lean control participants and athletes, and total and saturated IMTG localised in the nucleus had a significant inverse relationship with insulin sensitivity. Total cytosolic IMTG was not different between groups, but saturated cytosolic IMTG species were significantly increased in individuals with type 2 diabetes compared with all other groups. There were no significant differences between groups for IMTG concentration in the mitochondria/endoplasmic reticulum., Conclusions/interpretation: These data reveal previously unknown differences in subcellular IMTG localisation based on metabolic health status and indicate the influence of sarcolemmal and nuclear IMTG on insulin sensitivity. Additionally, these studies suggest saturated IMTG may be uniquely deleterious for muscle insulin sensitivity. Graphical abstract.

Coenzyme Q10 (CoQ 10 ) is an essential part of the mitochondrial respiratory chain . Here, we describe an accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of mitochondrial CoQ 10 in isolated mitochondria . In the assay, mitochondrial suspensions are spiked with CoQ 10 -[ 2 H 9 ] internal standard (IS), extracted with organic solvents and CoQ 10 quantified by LC-MS/MS using multiple reaction monitoring (MRM).

In people without lower extremity peripheral artery disease (PAD), mitochondrial DNA copy number declines with aging, and this decline is associated with declines in mitochondrial activity and functional performance. However, whether lower extremity ischemia is associated with lower mitochondrial DNA copy number and whether mitochondrial DNA copy number is associated with the degree of functional impairment in people with PAD is unknown. In people with and without PAD, age 65 years and older, we studied associations of the ankle-brachial index (ABI) with mitochondrial DNA copy number and associations of mitochondrial DNA copy number with functional impairment. Calf muscle biopsies were obtained from 34 participants with PAD (mean age: 73.5 years (SD 6.4), mean ABI: 0.67 (SD 0.15), mean 6-minute walk distance: 1191 feet (SD 223)) and 10 controls without PAD (mean age: 73.1 years (SD 4.7), mean ABI: 1.14 (SD 0.07), mean 6-minute walk distance: 1387 feet (SD 488)). Adjusting for age and sex, lower ABI values were associated with higher mitochondrial DNA copy number, measured in relative copy number (ABI<0.60: 914, ABI 0.60-0.90: 731, ABI 0.90-1.50: 593; p trend=0.016). The association of mitochondrial DNA copy number with the 6-minute walk distance and 4-meter walking velocity differed significantly between participants with versus without PAD ( p-value for interaction=0.001 and p=0.015, respectively). The correlation coefficient between mitochondrial DNA copy number and the 6-minute walk distance was 0.653 ( p=0.056) among people without PAD and -0.254 ( p=0.154) among people with PAD and ABI < 0.90. In conclusion, lower ABI values are associated with increased mitochondrial DNA copy number. Associations of mitochondrial DNA copy number with the 6-minute walk distance and 4-meter walking velocity significantly differed between people with versus without PAD, with stronger positive associations observed in people without PAD than in people with PAD. The cross-sectional and exploratory nature of the analyses precludes conclusions regarding causal inferences. ClinicalTrials.gov Identifier: NCT02246660.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

All eukaryotic cells have adapted the use of the calcium ion (Ca 2+ ) as a universal signaling element through the evolution of a toolkit of Ca 2+ sensor, buffer and effector proteins. Among these toolkit components, integral and peripheral proteins decorate biomembranes and coordinate the movement of Ca 2+ between compartments, sense these concentration changes and elicit physiological signals. These changes in compartmentalized Ca 2+ levels are not mutually exclusive as signals propagate between compartments. For example, agonist induced surface receptor stimulation can lead to transient increases in cytosolic Ca 2+ sourced from endoplasmic reticulum (ER) stores; the decrease in ER luminal Ca 2+ can subsequently signal the opening surface channels which permit the movement of Ca 2+ from the extracellular space to the cytosol. Remarkably, the minuscule compartments of mitochondria can function as significant cytosolic Ca 2+ sinks by taking up Ca 2+ in a coordinated manner. In non-excitable cells, inositol 1,4,5 trisphosphate receptors (IP 3 Rs) on the ER respond to surface receptor stimulation; stromal interaction molecules (STIMs) sense the ER luminal Ca 2+ depletion and activate surface Orai1 channels; surface Orai1 channels selectively permit the movement of Ca 2+ from the extracellular space to the cytosol; uptake of Ca 2+ into the matrix through the mitochondrial Ca 2+ uniporter (MCU) further shapes the cytosolic Ca 2+ levels. Recent structural elucidations of these key Ca 2+ toolkit components have improved our understanding of how they function to orchestrate precise cytosolic Ca 2+ levels for specific physiological responses. This chapter reviews the atomic-resolution structures of IP 3 R, STIM1, Orai1 and MCU elucidated by X-ray crystallography, electron microscopy and NMR and discusses the mechanisms underlying their biological functions in their respective compartments within the cell.

Understanding the relationship between physical exercise, reactive oxygen species, and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Heart and skeletal muscles have a high density of mitochondria with robust energetic demands, and mitochondria plasticity has an important role in both the cardiovascular system and skeletal muscle responses. The aim of this study was to investigate the influence of regular physical activity on the oxidation profiles of mitochondrial proteins from heart and tibialis anterior muscles. To this end, we used the mouse as animal model. Mice were divided into two groups: untrained and regularly trained. The carbonylated protein pattern was studied by two-dimensional gel electrophoresis followed by Western blot with anti-dinitrophenyl hydrazone antibodies. Mass spectrometry analysis allowed the identification of several different protein oxidation sites, including methionine, cysteine, proline, and leucine residues. A large number of oxidized proteins were found in both untrained and trained animals. Moreover, mitochondria from skeletal muscles and heart showed almost the same carbonylation pattern. Interestingly, exercise training seems to increase the carbonylation level mainly of mitochondrial proteins from skeletal muscle.

Mitochondrial bioenergetics has been implicated in a number of vital cellular and physiological phenomena, including aging, metabolism, and stress resistance. Heterogeneity of the mitochondrial membrane potential (Δψ), which is central to organismal bioenergetics, has been successfully measured via flow cytometry in whole cells but rarely in isolated mitochondria from large animal models. Similar studies in small animal models, such as Caenorhabditis elegans (C. elegans), are critical to our understanding of human health and disease but lack analytical methodologies. Here we report on new methodological developments that make it possible to investigate the heterogeneity of Δψ in C. elegans during development and in tissue-specific studies. The flow cytometry methodology described here required an improved collagenase-3-based mitochondrial isolation procedure and labeling of mitochondria with the ratiometric fluorescent probe JC-9. To demonstrate feasibility of tissue-specific studies, we used C. elegans strains expressing blue-fluorescent muscle-specific proteins, which enabled identification of muscle mitochondria among mitochondria from other tissues. This methodology made it possible to observe, for the first time, critical changes in Δψ during C. elegans larval development and provided direct evidence of the elevated bioenergetic status of muscle mitochondria relative to their counterparts in the rest of the organism. Further application of these methodologies can help tease apart bioenergetics and other biological complexities in C. elegans and other small animal models used to investigate human disease and aging.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 μg of mitochondria indicate that 1.0 μg is sufficient to measure respiration and that 5.0 μg provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

A 43-year-old female patient diagnosed with chronic progressive external ophthalmoplegia (CPEO) because of mitochondrial myopathy documented by muscle biopsy is presented. The chief complaints were represented by blepharoptosis and ophthalmoplegia. The muscle biopsy was evaluated by histology, using the appropriate histochemical and histoenzimological stains. Ragged red fibers with Gomori trichrome stain were seen, which showed cytochrome c oxydase deficiency and abnormal succinate dehydrogenase staining in around 20% of muscle fibres. Electron microscopy was also performed which demonstrated abnormal, hyperplastic, pleomorphic, and hypertrophic mitochondria, characterized by paracrystalline inclusions arranged in parallel rows ("parking-lot" inclusions), consisting of rectangular arrays of mitochondrial membranes in a linear or grid-like pattern. In conclusion, mitochondrial myopathy was definitely diagnosed. Although molecular analysis, which was subsequently carried out, failed to reveal mutations in the mitochondrial DNA or in selected nuclear genes, the pathologic diagnosis was not changed. The differential diagnosis of CPEO with other forms of ocular myopathies as well as the possible association of CPEO with systemic syndromes is discussed. Ophtalmologists and medical internists should always suspect CPEO when dealing with patients affected by ocular myopathy, either in its pure form or in association with other myopathic or systemic signs.

Background: We considered of interest to evaluate how aging affects mitochondrial function in skeletal muscle., Methods: We measured mitochondrial oxidative capacity and proton leak, together with lipid oxidative damage, superoxide dismutase specific activity and uncoupling protein 3 content, in subsarcolemmal and intermyofibrillar mitochondria from adult (six months) and old (two years) rats. Body composition, resting metabolic rate and plasma non esterified fatty acid levels were also assessed., Results: Old rats displayed significantly higher body energy and lipids, while body proteins were significantly lower, compared to adult rats. In addition, plasma non esterified fatty acid levels were significantly higher, while resting metabolic rates were found to be significantly lower, in old rats compared to adult ones. Significantly lower oxidative capacities in whole tissue homogenates and in intermyofibrillar and subsarcolemmal mitochondria were found in old rats compared to adult ones. Subsarcolemmal and intermyofibrillar mitochondria from old rats exhibited a significantly lower proton leak rate, while oxidative damage was found to be significantly higher only in subsarcolemmal mitochondria. Mitochondrial superoxide dismutase specific activity was not significantly affected in old rats, while significantly higher content of uncoupling protein 3 was found in both mitochondrial populations from old rats compared to adult ones, although the magnitude of the increase was lower in subsarcolemmal than in intermyofibrillar mitochondria., Conclusions: The decrease in oxidative capacity and proton leak in intermyofibrillar and subsarcolemmal mitochondria could induce a decline in energy expenditure and thus contribute to the reduced resting metabolic rate found in old rats, while oxidative damage is present only in subsarcolemmal mitochondria.

Mitochondrial alterations are the most common feature of human myopathies. A biopsy of quadriceps muscle from a 50-year-old woman exhibiting myopathic symptoms was examined by transmission electron microscopy. Biopsied fibers from quadriceps muscle displayed numerous subsarcolemmal mitochondria that contained crystalloids. Numbering 1-6 per organelle, these consisted of rows of punctuate densities measuring ∼0.34 nm; the parallel rows of these dots had a periodicity of ∼0.8 nm. The crystalloids were ensconced within cristae or in the outer compartment. Some mitochondria without crystalloids had circumferential cristae, leaving a membrane-free center that was filled with a farinaceous material. Other scattered fibrocyte defects included disruption of the contractile apparatus or its sporadic replacement by a finely punctuate material in some myofibers. Intramitochondrial crystalloids, although morphologically striking, do not impair organelle physiology to a significant degree, so the muscle weakness of the patient must originate elsewhere.

Background: While there is agreement that exercise is a powerful stimulus to increase both mitochondrial function and content, we do not know the optimal training stimulus to maximise improvements in mitochondrial biogenesis., Scope of Review: This review will focus predominantly on the effects of exercise on mitochondrial function and content, as there is a greater volume of published research on these adaptations and stronger conclusions can be made., Major Conclusions: The results of cross-sectional studies, as well as training studies involving rats and humans, suggest that training intensity may be an important determinant of improvements in mitochondrial function (as determined by mitochondrial respiration), but not mitochondrial content (as assessed by citrate synthase activity). In contrast, it appears that training volume, rather than training intensity, may be an important determinant of exercise-induced improvements in mitochondrial content. Exercise-induced mitochondrial adaptations are quickly reversed following a reduction or cessation of physical activity, highlighting that skeletal muscle is a remarkably plastic tissue. Due to the small number of studies, more research is required to verify the trends highlighted in this review, and further studies are required to investigate the effects of different types of training on the mitochondrial sub-populations and also mitochondrial adaptations in different fibre types. Further research is also required to better understand how genetic variants influence the large individual variability for exercise-induced changes in mitochondrial biogenesis., General Significance: The importance of mitochondria for both athletic performance and health underlines the importance of better understanding the factors that regulate exercise-induced changes in mitochondrial biogenesis. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research., (© 2013.)

Several lines of evidence indicate that reactive oxygen species (ROS) overproduction under the metabolic syndrome condition is the leading cause of cardiovascular events. Calcium is an important stimulus for vasoconstriction and plays a pivotal role in the development of hypertension. Here, we investigate whether a relationship exists between metabolic syndrome-induced mitochondrial ROS overproduction and Ang II-mediated Ca2+ release in vascular smooth muscle cells (VSMC). The effect of mitochondrial ROS on AT1 expression, and Ca2+ and IP3 generation was studied in 2 VSMC models of metabolic syndrome using fura-2/AM probes and ELISA-based assay. Ang II-mediated aortic ring contraction in SD rats fed with high-fat diet (HFD) was measured using a force transducer connected to chart recorder. In the metabolic syndrome, almost 2-fold increased mitochondrial O2 - significantly upregulated AT1 expressions by ~60%, companied by elevated Ca2+ and IP3 levels in VSMC and enhanced aortic rings contraction. All these increments were blocked by rotenone (inhibitor of mitochondrial respiratory chain complex I), ruthenium red (inhibitor of calcium uniporter), cyclosporin A (inhibitor of mitochondrial permeability pore), and N-acetylcysteine. Therefore, in the states of metabolic syndrome, ROS overproduction in mitochondrial complex I enhances Ang II-mediated vascular contraction via an AT1-dependent pathway. In addition, the import of Ca2+ from endoplasmic reticulum to mitochondria via calcium uniporter and mitochondrial permeability pore seems to serve as a mechanism to further aggravate mitochondrial damage and vascular dysfunction that may contribute to the occurrence of hypertension., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Mitochondrial dysfunction is an established feature of multiple sclerosis (MS). We recently described high levels of mitochondrial DNA (mtDNA) deletions within respiratory enzyme-deficient (lacking mitochondrial respiratory chain complex IV with intact complex II) neurons and choroid plexus epithelial cells in progressive MS., Objectives: The objective of this paper is to determine whether respiratory enzyme deficiency and mtDNA deletions in MS were in excess of age-related changes within muscle, which, like neurons, are post-mitotic cells that frequently harbour mtDNA deletions with ageing and in disease., Methods: In progressive MS cases (n=17), known to harbour an excess of mtDNA deletions in the central nervous system (CNS), and controls (n=15), we studied muscle (paraspinal) and explored mitochondria in single fibres. Histochemistry, immunohistochemistry, laser microdissection, real-time polymerase chain reaction (PCR), long-range PCR and sequencing were used to resolve the single muscle fibres., Results: The percentage of respiratory enzyme-deficient muscle fibres, mtDNA deletion level and percentage of muscle fibres harbouring high levels of mtDNA deletions were not significantly different in MS compared with controls., Conclusion: Our findings do not provide support to the existence of a diffuse mitochondrial abnormality involving multiple systems in MS. Understanding the cause(s) of the CNS mitochondrial dysfunction in progressive MS remains a research priority.

Mitochondrial density in skeletal muscle fibers is governed by the demand for aerobic ATP production, but the heterogeneous distribution of these mitochondria appears to be governed by constraints associated with oxygen diffusion. We propose that each muscle fiber has an optimal mitochondrial distribution at which it attains a near maximal rate of ATP consumption (RATPase ) while mitochondria are exposed to a minimal oxygen concentration, thus minimizing reactive oxygen species (ROS) production. We developed a coupled reaction-diffusion/cellular automata (CA) mathematical model of mitochondrial function and considered four fiber types in mouse extensor digitorum longus (EDL) and soleus (SOL) muscle. The developed mathematical model uses a reaction-diffusion analysis of metabolites including oxygen, ATP, ADP, phosphate, and phosphocreatine (PCr) involved in energy metabolism and mitochondrial function. A CA approach governing mitochondrial life cycles in response to the metabolic state of the fiber was superimposed and coupled to the reaction-diffusion approach. The model results show the sensitivity of important model outputs such as the RATPase , effectiveness factor (η) and average oxygen concentration available at each mitochondrion to local oxygen concentration in the fibers through variation in the CA model parameter θdet , which defines the sensitivity of mitochondrial death to the oxygen concentration. The predicted optimal mitochondrial distributions matched previous experimental findings. Deviations from this optimal distribution corresponding to higher CA model parameter values (a more uniform mitochondrial distribution) lead to lower aerobic rates. In contrast, distributions corresponding to lower CA model parameter values (a more asymmetric distribution) lead to an increased exposure of mitochondria to oxygen, usually without substantial increases in aerobic rates, which would presumably result in increased ROS production and thus increased risks of cytotoxicity., (Copyright © 2013 Wiley Periodicals, Inc.)

Uncoupling proteins (UCP) are able to increase H(+) leakage across the inner mitochondrial membrane, thus dissipating the membrane potential and increasing oxygen consumption. Despite the identification of several UCP orthologs in birds, reptiles, amphibians and fish, little is known about their functional properties in fish. The aim of this work was to identify and characterize a UCP in mitochondria found in goldfish white skeletal muscle. Western blot analysis, using a polyclonal antibody raised against mammalian UCP3, showed a single band at approximately 32 kDa. During non-phosphorylating respiration, we observed that palmitate promoted a dose-dependent increase in oxygen consumption that is abolished by addition of BSA (fatty acid chelator). Interestingly, this palmitate-induced increase in oxygen consumption was not inhibited by GDP, a well-known UCP inhibitor. In phosphorylating mitochondria, palmitate lowered both ADP/O ratio (number of atoms of phosphorus incorporated as ATP per molecule of O2 consumed) and the respiratory control ratio. Moreover, we found that different fatty acids can modulate mitochondrial membrane potential. In conclusion, our results suggest that goldfish UCP is functionally similar to the UCP found in other species, including mammals.

Intramyocellular lipid (IMCL) is predominantly stored as intramuscular triglyceride (IMTG) in lipid droplets and is utilized as metabolic fuel during physical exercise. IMTG is also implicated in muscle insulin resistance (IR) in type 2 diabetes. However, it has become apparent that lipid moieties such as ceramide and diacylglycerol are the likely culprits of IR. This article reviews current knowledge of IMCL-mediated IR and important areas of investigation, including myocellular lipid transport and lipid droplet proteins. Several crucial questions remain unanswered, such as the identity of specific ceramide and diacylglycerol species that mediate IR in human muscle and their subcellular location. Quantitative lipidomics and proteomics of targeted subcellular organelles will help to better define the mechanisms underlying pathological IMCL accumulation and IR., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Skeletal muscle mitochondrial content varies extensively between human subjects. Biochemical measures of mitochondrial proteins, enzyme activities and lipids are often used as markers of mitochondrial content and muscle oxidative capacity (OXPHOS). The purpose of this study was to determine how closely associated these commonly used biochemical measures are to muscle mitochondrial content and OXPHOS. Sixteen young healthy male subjects were recruited for this study. Subjects completed a graded exercise test to determine maximal oxygen uptake (VO2peak) and muscle biopsies were obtained from the vastus lateralis. Mitochondrial content was determined using transmission electron microscopy imaging and OXPHOS was determined as the maximal coupled respiration in permeabilized fibres. Biomarkers of interest were citrate synthase (CS) activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I–V protein content, and complex I–IV activity. Spearman correlation coefficient tests and Lin's concordance tests were applied to assess the absolute and relative association between the markers and mitochondrial content or OXPHOS. Subjects had a large range of VO2peak (range 29.9–71.6ml min−1 kg−1) and mitochondrial content (4–15% of cell volume).Cardiolipin content showed the strongest association with mitochondrial content followed by CS and complex I activities. mtDNA was not related to mitochondrial content. Complex IV activity showed the strongest association with muscle oxidative capacity followed by complex II activity.We conclude that cardiolipin content, and CS and complex I activities are the biomarkers that exhibit the strongest association with mitochondrial content, while complex IV activity is strongly associated with OXPHOS capacity in human skeletal muscle.

Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance and type 2 diabetes. Considering the importance of mitochondrial dynamics in mitochondrial and cellular functions, we hypothesized that obesity and excess energy intake shift the balance of mitochondrial dynamics, further contributing to mitochondrial dysfunction and metabolic deterioration in skeletal muscle. First, we revealed that excess palmitate (PA), but not hyperglycemia, hyperinsulinemia, or elevated tumor necrosis factor alpha, induced mitochondrial fragmentation and increased mitochondrion-associated Drp1 and Fis1 in differentiated C2C12 muscle cells. This fragmentation was associated with increased oxidative stress, mitochondrial depolarization, loss of ATP production, and reduced insulin-stimulated glucose uptake. Both genetic and pharmacological inhibition of Drp1 attenuated PA-induced mitochondrial fragmentation, mitochondrial depolarization, and insulin resistance in C2C12 cells. Furthermore, we found smaller and shorter mitochondria and increased mitochondrial fission machinery in the skeletal muscle of mice with genetic obesity and those with diet-induced obesity. Inhibition of mitochondrial fission improved the muscle insulin signaling and systemic insulin sensitivity of obese mice. Our findings indicated that aberrant mitochondrial fission is causally associated with mitochondrial dysfunction and insulin resistance in skeletal muscle. Thus, disruption of mitochondrial dynamics may underlie the pathogenesis of muscle insulin resistance in obesity and type 2 diabetes.

Red and white muscles are faced with very different energetic demands. However, it is unclear whether relative mitochondrial protein expression is different between muscle types. Mitochondria from red and white porcine skeletal muscle were isolated with a Percoll gradient. Differences in protein composition were determined using blue native (BN)-PAGE, two-dimensional differential in gel electrophoresis (2D DIGE), optical spectroscopy, and isobaric tag for relative and absolute quantitation (iTRAQ). Complex IV and V activities were compared using BN-PAGE in-gel activity assays, and maximal mitochondrial respiration rates were assessed using pyruvate (P) + malate (M), glutamate (G) + M, and palmitoyl-carnitine (PC) + M. Without the Percoll step, major cytosolic protein contamination was noted for white mitochondria. Upon removal of contamination, very few protein differences were observed between red and white mitochondria. BN-PAGE showed no differences in the subunit composition of Complexes I-V or the activities of Complexes IV and V. iTRAQ analysis detected 358 mitochondrial proteins, 69 statistically different. Physiological significance may be lower: at a 25% difference, 48 proteins were detected; at 50%, 14 proteins were detected; and 3 proteins were detected at a 100%. Thus any changes could be argued to be physiologically modest. One area of difference was fat metabolism where four β-oxidation enzymes were ∼25% higher in red mitochondria. This was correlated with a 40% higher rate of PC+M oxidation in red mitochondria compared with white mitochondria with no differences in P+M and G+M oxidation. These data suggest that metabolic demand differences between red and white muscle fibers are primarily matched by the number of mitochondria and not by significant alterations in the mitochondria themselves.

Adaptation of skeletal muscle to repeated bouts of endurance exercise increases aerobic capacity and improves mitochondrial function. However, the adaptation of human skeletal muscle mitochondrial proteome to short-term endurance exercise training has not been investigated. Eight sedentary males cycled for 60 min at 80% of peak oxygen consumption (VO(2peak) ) each day for 14 consecutive days, resulting in an increase in VO(2peak) of 17.5±3.8% (p<0.01). Mitochondria-enriched protein fractions from skeletal muscle biopsies taken from m. vastus lateralis at baseline, and on the morning following the 7th and 14th training sessions were subjected to 2-D DIGE analysis with subsequent MS followed by database interrogation to identify the proteins of interest. Thirty-one protein spots were differentially expressed after either 7 or 14 days of training (ANOVA, p<0.05). These proteins included subunits of the electron transport chain, enzymes of the tricarboxylic acid cycle, phosphotransfer enzymes, and regulatory factors in mitochondrial protein synthesis, oxygen transport, and antioxidant capacity. Several proteins demonstrated a time course-dependent induction during training. Our results illustrate the phenomenon of skeletal muscle plasticity with the extensive remodelling of the mitochondrial proteome occurring after just 7 days of exercise training suggestive of enhanced capacity for adenosine triphosphate generation at a cellular level., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles. Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37°C⁷. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 μg) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5 mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 μM) is added to start state 3. Oligomycin (1 μM), an ATPase synthase blocker, is used to estimate state. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 μM) is added to measurestate, or uncoupled respiration. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR ≥ 4 is considered as evidence of a viable mitochondria preparation. In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

Mitochondria are highly heterogeneous organelles that likely have unique isoelectric points (pI), which are related to their surface compositions and could be exploited in their purification and isolation. Previous methods to determine pI of mitochondria report an average pI. This article is the first report of the determination of the isoelectric points of individual mitochondria by capillary isoelectric focusing (cIEF). In this method, mitochondria labeled with the mitochondrial-specific probe 10-N-nonyl acridine orange (NAO) are injected into a fused-silica capillary in a solution of carrier ampholytes at physiological pH and osmolarity, where they are focused then chemically mobilized and detected by laser-induced fluorescence (LIF). Fluorescein-derived pI markers are used as internal standards to assign a pI value to each individually detected mitochondrial event, and a mitochondrial pI distribution is determined. This method provides reproducible distributions of individual mitochondrial pI, accurate determination of the pI of individual mitochondria by the use of internal standards, and resolution of 0.03 pH units between individual mitochondria. This method could also be applied to investigate or design separations of organelle subtypes (e.g., subsarcolemmal and interfibrillar skeletal muscle mitochondria) and to determine the pIs of other biological or nonbiological particles.

Reactive Oxygen Species (ROS) constitute important intracellular signaling molecules. Mitochondria are admitted sources of ROS, especially of superoxide anions through the electron transport chain. Here the mitochondria-targeted ratiometric pericam (RPmt) was used as a superoxide biosensor, by appropriate choice of the excitation wavelength. RPmt was transfected in vivo into mouse muscles. Confocal imaging of isolated muscle fibers reveals spontaneous flashes of RPmt fluorescence. Flashes correspond to increases in superoxide production, as shown by simultaneous recordings of the fluorescence from MitoSox, a mitochondrial superoxide probe. Flashes occur in all subcellular populations of mitochondria. Spatial analysis of the flashes pattern over time revealed that arrays of mitochondria work as well-defined superoxide-production-units. Increase of superoxide production at the muscle fiber level involves recruitment of supplemental units with no increase in per-unit production. Altogether, these results demonstrate that superoxide flashes in muscle fibers correspond to physiological signals linked to mitochondrial metabolism. They also suggest that superoxide, or one of its derivatives, modulates its own production at the mitochondrial level.

The aim of this study was to investigate the influence of lifestyle on the aging alterations in skeletal muscle mitochondrial proteins. Thirty C57BL/6 strain mice (2 months) were randomly divided into three groups (young, Y; old sedentary, S; and old active, A). The S and A mice were individually placed into standard cages and in cages with running wheels for 25 months. Upon killing, mitochondria from the hind limb skeletal muscles were isolated for the evaluation of general proteome alterations, carbonylation, and electron transport chain (ETC) activity. We identified 77 different proteins mostly from the oxidative phosphorylation and mitochondrial metabolism. Sedentary mice presented a significant loss of ETC functionality in opposition to active mice. Although some proteins were found damaged in both A and S mice, damage to ETC proteins was more evident in S. Moreover, it is also possible to conclude that lifestyle is a key modulator for preventing the aging-induced protein expression and functionality in mitochondria.

The production of H(2)O(2) by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H(2)O(2). However, matrix enzymes such as the glutathione peroxidases can consume H(2)O(2) and compete with efflux of H(2)O(2), causing an underestimation of superoxide production. To assess this underestimate, we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90% as a consequence of pretreatment with 1-chloro-2,4-dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H(2)O(2), consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H(2)O(2) formation from matrix-directed superoxide by up to two-fold from several sites of production, as defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.2-2.5 nmol H(2)O(2) min(-1) mg protein(-1). Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H(2)O(2) efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H(2)O(2) efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: CDNB-pretreated rate = control rate + [1.43 x (control rate)]/(0.55 + control rate). These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria.

The organ most frequently affected in mitochondrial disorders is the skeletal muscle (mitochondrial myopathy). Mitochondrial myopathies may be part of syndromic as well as non-syndromic mitochondrial disorders. Involvement of the skeletal muscle may remain subclinical, may manifest as isolated elevation of the creatine-kinase, or as weakness and wasting of one or several muscle groups. The course of mitochondrial myopathies is usually slowly progressive and only rarely rapidly progressive leading to restriction of mobility and requirement of a wheel chair or even muscular respiratory insufficiency. Frequently reported symptoms of mitochondrial myopathies are permanent tiredness, easy fatigability, muscle aching at rest or already after moderate exercise, muscle cramps, muscle stiffness, fasciculations and muscle weakness. The diagnosis is based on the history, clinical neurologic examination, blood chemical investigations, lactate stress test, electromyography, magnetic resonance imaging, magnetic resonance spectroscopy, muscle biopsy, biochemical investigations of the skeletal muscles, and genetic investigations. Only symptomatic therapy is available and includes physiotherapy and orthopedic supportive devices, diet, symptomatic drug therapy (analgetics, cramp-releasing drugs, antioxidants, lactate-lowering drugs, alternative energy sources, co-factors), avoidance of mitochondrion-toxic drugs, surgery (correction of ptosis or orthopedic problems), and invasive or non-invasive mechanical ventilation. General anesthesia needs to be performed in the same way as in patients with susceptibility for malignant hyperthermia., (Georg Thieme Verlag KG Stuttgart, New York.)

Changes in membrane lipid composition (membrane remodelling) have been associated with metabolic depression in some aestivating snails but has not been studied in aestivating frogs. This study examined the membrane phospholipid composition of two Australian aestivating frog species Cyclorana alboguttata and Cyclorana australis. The results showed no major membrane remodelling of tissue in either frog species, or in mitochondria of C. alboguttata due to aestivation. Mitochondrial membrane remodelling was not investigated in C. australis. Where investigated in C. alboguttata, total protein and phospholipid content, and citrate synthase (CS) and cytochrome c oxidase (CCO) activities in tissues and mitochondria mostly did not change with aestivation in liver. In skeletal muscle, however, CS and CCO activities, mitochondrial and tissue phospholipids, and mitochondrial protein decreased with aestivation. These decreases in muscle indicate that skeletal muscle mitochondrial content may decrease during aestivation. Na(+)K(+)ATPase activity of both frog species showed no effect of aestivation. In C. alboguttata different fat diets had a major effect on both tissue and mitochondrial phospholipid composition indicating an ability to remodel membrane composition that is not utilised in aestivation. Therefore, changes in lipid composition associated with some aestivating snails do not occur during aestivation in these Australian frogs.

Mitochondrial DNA (mt-DNA) disorders and abnormal regulation of nuclear-derived proteins devoted to the cross-talk between the two cellular genomes have recently interested researchers in the field of neuromuscular diseases. We have identified, isolated and sequenced a new gene, augmenter of liver regeneration (ALR) that stimulates in vivo hepatocyte proliferation and up-regulates mt-DNA expression and ATP production. ALR protein (Alrp) is mainly located, in rat, in the mitochondrial inter-membrane space and its mRNA is particularly abundant in brain, muscle, testis and liver, tissues whose activity is mostly dependent on mitochondrial metabolism. Studies on rat Alrp sequence revealed the presence of homologous amino-acid sections into proteins derived from mouse, human, Drosophyla, plants and even DNA viruses. In this article, we evaluated ALR expression in normal human muscular tissues, both as protein and as mRNA. The data, obtained by molecular biology, immunohistochemistry and electron microscopy, demonstrated that: (i) Alrp and ALR mRNA are present in human muscular tissue; (ii) Alrp is particularly expressed in muscular fibres rich in mitochondria; (iii) Alrp is localized in the mitochondrial inter-membrane space or associated to mitochondrial cristae; and (iv) in subjects younger then 35 years of age, ALR mRNA expression is different between male and female subjects. In conclusion, the present data set Alrp, as a factor associated with mitochondria also in human tissue, call for future studies aimed at establishing Alrp as an important factor involved in the molecular events that trigger neuromuscular diseases.

The increase in the levels of protein carbonyls, biomarkers of oxidative stress, appears to play an important role in aging skeletal muscle. However, the exact distributions of carbonyls among various skeletal muscle microstructures still remain largely unknown, partly owing to the lack of adequate techniques to carry out these measurements. This report describes an immunohistochemical approach to determine the relative abundance of carbonyls in the intermyofibrillar mitochondria (IFM), the subsarcolemmal mitochondria (SSM), the cytoplasm, and the extracellular space of skeletal muscle. These morphological features were defined by labeling the nucleus, the Z-lines, and mitochondria. Carbonyls were detected by derivatization with dinitrophenylhydrazine followed by labeling with an Alexa 488-labeled anti-dinitrophenyl primary antibody. Alexa 488 fluorescence (green) in different fiber microstructures was used to estimate the relative abundance of carbonyls. On the basis of the samples examined, preliminary results suggest that the most dramatic age-related changes in carbonyl levels occur in the extracellular space, followed in a decreasing order by SSM, IFM, and the cytoplasm. These observations were confirmed in the soleus and semimembranosus muscles composed predominantly of type I and type II fibers, respectively. This approach could easily be extended to the investigation of carbonyl levels in other muscles (composed of mixed skeletal muscle fiber types) or other tissues in which protein carbonyls are present.

Carbonyl-modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE-LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ) is used for protein labeling. BSA subjected to metal-catalyzed oxidation is used to optimize the labeling reactions, confirm the separation power of CSE, and characterize the response of the LIF detector. The method is capable of detecting femtomole (fmol) amounts of carbonyls in proteins with molecular masses ranging from 26 to 30 kDa. Using this method, we determined that mitochondrial proteins isolated from skeletal muscle contains 2.1 +/- 0.1 (average +/- SD; n = 3) nmol carbonyl/mg protein. The methodology described here should be compatible with the analysis of single cells and needle biopsies taken from oxidative stress animal models.

The objective of the present study was to develop a combination of fluorescent stains that would allow visualisation of the network of mitochondria and lipid droplets (intramyocellular lipids or IMCL) in human skeletal muscle fibres by means of conventional and confocal microscopy. Muscle biopsies were taken from the vastus lateralis of three lean, healthy and physically active male subjects. Frozen muscle sections were stained for mitochondria using antibodies against three mitochondrial proteins; porin, cytochrome c oxidase (COX) and NADH-ubiquinol oxidoreductase and neutral lipids were stained with oil red O. Anti-COX staining produced images with the strongest fluorescence signal and the highest resolution of the mitochondrial network and this stain was successfully combined with the antibody against type I fibre myosin. A highly organised matrix arrangement of mitochondria within the sarcomeres (in pairs at the I-band) was observed in the oxidative type I fibres. The density of mitochondria was the highest in the subsarcolemmal region. Anti-COX staining was combined with oil red O demonstrating that in type I fibres lipid droplets are mainly located in the space between the mitochondria.

Cardiac and diaphragmatic mitochondria from male SAMP8 (senescent) and SAMR1 (resistant) mice of 5 or 10 months of age were studied. Levels of lipid peroxidation (LPO), glutathione (GSH), GSH disulfide (GSSG), and GSH peroxidase and GSH reductase (GRd) activities were measured. In addition, the effect of chronic treatment with the antioxidant melatonin from 1 to 10 months of age was evaluated. Cardiac and diaphragmatic mitochondria show an age-dependent increase in LPO levels and a reduction in GSH:GSSG ratios. Chronic treatment with melatonin counteracted the age-dependent LPO increase and GSH:GSSG ratio reduction in these mitochondria. Melatonin also increased GRd activity, an effect that may account for the maintenance of the mitochondrial GSH pool. Total mitochondrial content of GSH increased after melatonin treatment. In general, the effects of age and melatonin treatment were similar in senescence-resistant mice (SAMR1) and SAMP8 cardiac and diaphragmatic mitochondria, suggesting that these mice strains display similar mitochondrial oxidative damage at the age of 10 months. The results also support the efficacy of long-term melatonin treatment in preventing the age-dependent mitochondrial oxidative stress.

We describe a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin hydrazide labeling of protein carbonyl groups, avidin affinity chromatography, multiplexed iTRAQ reagent stable isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer. This strategy provided the ability to distinguish biotin hydrazide labeled, avidin purified, carbonylated proteins from non-carbonylated background proteins with affinity for the avidin column, derived from a control sample. Applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, we generated a catalogue of over 200 carbonylated proteins by virtue of their quantitative enrichment compared to the control sample. The catalogue contains many mitochondrial localized proteins shown to be susceptible to carbonyl modification for the first time, including numerous transmembrane proteins involved in oxidative phosphorylation. Other oxidative modifications (e.g. nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, providing further evidence of the susceptibility of these proteins to oxidative damage. The results also demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent-labeled peptide mixtures, as well as the quantitative reproducibility of the avidin-affinity enrichment method.

The present study determined if acute exercise increased free radical formation in human skeletal muscle. Vastus lateralis biopsies were obtained in a randomized balanced order from six males at rest and following single-leg knee extensor exercise performed for 2 min at 50% of maximal work rate (WR(MAX)) and 3 min at 100% WR(MAX). EPR spectroscopy revealed an exercise-induced increase in mitochondrial ubisemiquinone (UQ*-) [0.167 +/- 0.055 vs. rest: 0.106 +/- 0.047 arbitrary units (AU)/g total protein (TP), P < 0.05] and alpha-phenyl-tert-butylnitrone-adducts (112 +/- 41 vs. rest: 29 +/- 9 AU/mg tissue mass, P < 0.05). Intramuscular lipid hydroperoxides also increased (0.320 +/- 0.263 vs. rest: 0.148 +/- 0.071 nmol/mg TP, P < 0.05) despite an uptake of alpha-tocopherol, alpha-carotene and beta-carotene. There were no relationships between mitochondrial volume density and any biomarkers of oxidative stress. These findings provide the first direct evidence for intramuscular free radical accumulation and lipid peroxidation following acute exercise in humans.

Changes in the properties of mitochondria from oxidative muscle of rainbow trout Oncorhynchus mykiss were examined during warm (5 degrees C to 15 degrees C) acclimation. Trout were studied shortly after the initial thermal change and after 8 weeks acclimation to 15 degrees C. To identify potential mechanisms by which oxidative capacities change, the modifications of phospholipid composition, membrane proteins and functional capacities of red muscle mitochondria were examined. Marked functional changes of isolated muscle mitochondria during warm acclimation of rainbow trout were reflected by a host of modifications in phospholipid composition, but by few shifts in protein components. Shortly after transfer of trout from 5 degrees C to 15 degrees C, the maximal oxidative capacity of mitochondria measured at 15 degrees C increased slightly, but rates at both assay temperatures (5 degrees C and 15 degrees C) decreased markedly after warm acclimation. The increase in capacity in short-term warm exposed trout was most pronounced when rates at 15 degrees C were expressed relative to cytochrome a and c(1) levels. Non-phosphorylating (State 4) rates of oxygen uptake increased with short-term warm exposure before returning to initial levels after warm acclimation. Cytochrome c oxidase (CCO) activity in the mitochondrial preparations decreased with warm acclimation. The thermal sensitivity of the ADP affinity was markedly modified during short-term warm exposure, when the ADP/O ratio increased, but warm acclimation returned these values to those observed initially. ADP affinity increased after warm acclimation. Changes in the mitochondrial content of cytochromes and adenine nucleotide translocase (ANT) could not explain these patterns. On the other hand, changes in the proportions of the lipid classes and in the acyl chain composition of certain phospholipid classes mirror the modifications in functional properties. Short-term exposure to 15 degrees C decreased the ratio of diacylphosphatidylethanolamine/diacylphosphatidylcholine (diacylPE/diacylPC), whereas warm acclimation led to restructuring of fatty acids (FA) and to increases of plasmalogen forms of PE and PC. Modification of overall membrane unsaturation did not appear to be the primary aim of restructuring membrane FA during warm acclimation, as total mitochondrial phospholipids and the major phospholipid classes only showed slight shifts of their acyl composition with warm acclimation. On the other hand, natural lysophosphatidylcholine (LysoPC) showed dramatic changes in FA content, as 16:0 and 18:1n-9 doubled whereas 22:6n-3 decreased from around 50% to 32% in warm acclimated trout. Similarly, in cardiolipin (CL), the levels of 16:0 and 18:1n-7 halved while 18:2n-6 increased to over 20% of the FA with warm acclimation. Given the central role of CL in modulating the activity of CCO, F(0)F(1)-ATPase and ANT, these changes suggest that specific compositional changes in CL are important modulators of mitochondrial capacities. The many structural changes in membrane lipids contrast with the limited modifications of the membrane protein components examined and support the concept of lipid structure modulating mitochondrial capacities.

Capillary electrophoresis (CE) with postcolumn laser-induced fluorescence detection (LIF) was used to analyze single skeletal muscle fibers from young and old rats. Due to selective labeling of mitochondria with 10-N-nonyl acridine orange, the zeptomole (10(-21) mole) sensitivity, and the high separation power, three properties of individual mitochondrial particles were revealed: the number, the distributions of cardiolipin, and their electrophoretic mobilities. Type I fibers had more mitochondrial particles and cardiolipin per particle than did type IIb fibers from rats of similar age. Individual fibers of the same fiber type from young rats contained more mitochondrial particles and cardiolipin per particle than did fibers from old rats. There were fiber type-specific and age-specific differences in the electrophoretic mobility of individual mitochondrial particles. The CE-LIF results of individual mitochondrial particles are the first of their kind in that they reveal fiber type-specific and age-specific differences that are not obviously noticed in bulk measurements of heterogeneous tissues.

Objectives: To evaluate the changes in the mitochondrial ATP production during sepsis and the participation of iNOS in these changes. We also assessed the effect of melatonin administration in this experimental paradigm., Methods: The activity of ATPase, the level of adenine nucleotides, and the ATP production were measured in mitochondria of diaphragm and hind leg skeletal muscle of wild type (iNOS+/+) and knockout iNOS (iNOS-/-) mice. Three experimental groups were done: control group; group of septic mice induced by cecal ligation and puncture (CLP), and group of septic mice treated with melatonin. Mice were killed 24 hours after CLP. Melatonin was administrated in four doses (30 mg/kg b.w.) as follows: 30 min before CLP (i.p.) and 30 min, 4 h and 8 h after CLP (s.c.)., Results: Mitochondrial production of ATP decreased in iNOS+/+ but not in iNOS-/- mice after sepsis. No changes in the ATPase activity were found in any group. Melatonin treatment normalized the production of ATP in iNOS+/+ mice, without affecting iNOS-/- animals., Conclusions: The reduction of the ATP production in iNOS+/+ but not in iNOS-/- mice suggest the participation of iNOS in the impairment of mitochondrial function in the former. Because ATPase was unaffected by sepsis, it is suggested the ATP deficit depended on the sepsis-induced respiratory chain damage. The normalization of the production of ATP with melatonin may explain the reduction of the mortality reported elsewhere in experimental and clinical sepsis after treatment with the indoleamine.

The fatty acid composition of phospholipids is an important determinant of membrane function. Although the mitochondria play a pivotal role in skeletal muscle function, the fatty acid composition of their individual phospholipids has not been examined. The purpose of this study was to determine the fatty acid profile of each phospholipid in rat skeletal muscle mitochondria and compare it with that of the whole muscle. Lipids were extracted from the gastrocnemius muscles of 10 Wistar rats, and phospholipids were separated by thin-layer chromatography. The fatty acid composition of each phospholipid was then determined by gas chromatography. The same procedure was applied to a mitochondrial preparation from these muscles. We found that the fatty acid composition of the individual mitochondrial phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, cardiolipin, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, and lysophosphatidyl choline) and of the total mitochondrial phospholipids differed markedly (P < 0.05) from the fatty acid composition of the corresponding whole muscle phospholipids. Notably, the mitochondrial phospholipids had higher percentages of MUFA [13.9 (2.1) vs. 10.3 (0.9)] and lower percentages of PUFA [34.8 (4.3) vs. 39.5 (5.2)] and n6 fatty acids [25.0 (2.5) vs. 27.6 (2.5)]. Overall, the mitochondrial phospholipids had a lower unsaturation index than whole muscle phospholipids [135 (20) vs. 161 (26)]. Because PUFA are susceptible to peroxidation, unlike saturated fatty acids and MUFA, we propose that the low polyunsaturation of mitochondrial phospholipids is the result of selective pressure toward membranes that are more resistant to oxidative damage by reactive oxygen species produced in their vicinity. The negative effect of the low polyunsaturation on membrane fluidity may be counterbalanced by the higher percentage of MUFA and the known low cholesterol content of mitochondrial membranes.

An accelerated rate of fat recovery (catch-up fat) and insulin resistance are characteristic features of weight recovery after caloric restriction, with implications for the pathophysiology of catch-up growth and weight fluctuations. Using a previously described rat model of weight recovery in which catch-up fat and skeletal muscle insulin resistance have been linked to suppressed thermogenesis per se, we investigated alterations in mitochondrial energetics and oxidative stress in subsarcolemmal (SS) and intermyofibrillar (IMF) skeletal muscle mitochondria. After 2 weeks of semistarvation followed by 1 week of refeeding, the refed rats show persistent and selective reductions in SS mitochondrial mass (assessed from citrate synthase activity in tissue homogenate and isolated mitochondria) and oxidative capacity. Furthermore, the refed rats show, in both SS and IMF muscle mitochondria, a lower aconitase activity (whose inactivation is an index of increased reactive oxygen species [ROS]), associated with higher superoxide dismutase activity and increased proton leak. Taken together, these studies suggest that diminished skeletal muscle mitochondrial mass and function, specifically in the SS mitochondrial compartment, contribute to the high metabolic efficiency for catch-up fat after caloric restriction and underscore a potential link between diminished skeletal muscle SS mitochondrial energetics, increased ROS concentration, and insulin resistance during catch-up fat.

Uncoupling protein (UCP) 1 (UCP1) catalyzes a proton leak in brown adipose tissue (BAT) mitochondria that results in nonshivering thermogenesis (NST), but the extent to which UCP homologs mediate NST in other tissues is controversial. To clarify the role of UCP3 in mediating NST in a hibernating species, we measured Ucp3 expression in skeletal muscle of arctic ground squirrels in one of three activity states (not hibernating, not hibernating and fasted for 48 h, or hibernating) and housed at 5 degrees C or -10 degrees C. We then compared Ucp3 mRNA levels in skeletal muscle with Ucp1 mRNA and UCP1 protein levels in BAT in the same animals. Ucp1 mRNA and UCP1 protein levels were increased on cold exposure and decreased with fasting, with the highest UCP1 levels in thermogenic hibernators. In contrast, Ucp3 mRNA levels were not affected by temperature but were increased 10-fold during fasting and >3-fold during hibernation. UCP3 protein levels were increased nearly fivefold in skeletal muscle mitochondria isolated from fasted squirrels compared with nonhibernators, but proton leak kinetics in the presence of BSA were unchanged. Proton leak in BAT mitochondria also did not differ between fed and fasted animals but did show classical inhibition by the purine nucleotide GDP. Levels of nonesterified fatty acids were highest during hibernation, and tissue temperatures during hibernation were related to Ucp1, but not Ucp3, expression. Taken together, these results do not support a role for UCP3 as a physiologically relevant mediator of NST in muscle.

Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver tissue sources. Indeed approximately one-third of the proteins identified in the soluble fractions are associated predominantly to one of the three tissues, indicating a tissue-dependent regulation of mitochondrial proteins. Furthermore a small percentage of the mitochondrial proteome is unique to each tissue.

This review discusses the present knowledge on the oxygen uptake kinetics at the onset of exercise in skeletal muscle and the contribution of a previously developed computer model of oxidative phosphorylation in intact skeletal muscle to the understanding of the factors determining this kinetics on the biochemical level. It has been demonstrated recently that an increase in the total creatine pool [PCr + Cr] and in glycolytic ATP supply lengthen the half-transition time of the VO2 on-kinetics, while an increase in mitochondria content, in parallel activation of ATP supply and ATP usage, in muscle oxygen concentration, in proton leak, in resting energy demand, in resting cytosolic pH, and in initial alkalization diminish this parameter. It has also been shown that the half-transition time is near-linearly proportional to the absolute difference between the phosphocreatine concentration during work and at rest (deltaPCr). The present review discusses whether the V/O2 on-kinetics on the muscle level is strictly or only approximately exponential. Finally, it is postulated that a short transition time of the VO2 on-kinetics in itself does not need be profitable for the skeletal muscle functioning during exercise, but usually a short transition time is correlated with factors that improve exercise capacity. The transition time is a phenomenological parameter resulting from the biochemical properties of the system and not a physical factor that can cause anything in the system.

We studied the molecular composition of muscle mitochondria to evaluate whether the contents of cytochromes or adenine nucleotide translocase (ANT) or phospholipid acyl compositions reflect differences in mitochondrial oxidative capacities. We isolated mitochondria from three vertebrates of similar size and preferred temperature, the rat (Rattus norvegicus), the cane toad (Bufo marinus) and the bearded dragon lizard (Pogona vitticeps). Mitochondrial oxidative capacities were higher in rats and cane toads than in bearded dragon, whether rates were expressed relative to protein, cytochromes or ANT. Inter-specific differences were least pronounced when rates were expressed relative to cytochrome A, a component of cytochrome C oxidase (CCO), or ANT. In mitochondria from rat and cane toad, cytochrome A was more abundant than C followed by B and then C(1), while in bearded dragon mitochondria, the cytochromes were present in roughly equal levels. Analysis of correlations between mitochondrial oxidative capacities and macromolecular components revealed that cytochrome A explained at least half of the intra- and inter-specific variability in substrate oxidation rates. ANT levels were an excellent correlate of state 3 rates while phospholipid contents were correlated with state 4 rates. As the % poly-unsaturation and the % 20:4n-6 in mitochondrial phospholipids were equivalent in toads and rats, and exceeded the levels in lizards, they may contribute to the inter-specific differences in oxidative capacities. We suggest that the numbers of CCO and ANT together with the poly-unsaturation of phospholipids explain the higher oxidative capacities in muscle mitochondria from rats and cane toads.

Previous findings suggested specific mitochondrial dysfunction in skeletal muscle of patients with amyotrophic lateral sclerosis (ALS). To answer the question of whether the dysfunction is specific, we investigated the histochemical distribution of mitochondrial marker activities, the ratio of mitochondrial (mt) versus nuclear (n) DNA, and the activities of citrate synthase (CS) and respiratory chain enzymes in muscle biopsies of 24 patients with sporadic ALS. The data were compared with those in 23 patients with other neurogenic atrophies (NAs), and 21 healthy controls. Muscle histology revealed similar signs of focally diminished mitochondrial oxidation activity in muscle fibres in both diseased groups. There was only minimal decline of mt/nDNA ratios in ALS and NA patients in comparison with healthy controls. The specific activities of mitochondrial markers CS and succinate dehydrogenase were significantly increased in both ALS and NA patients. The specific activities of respiratory chain enzymes were not significantly different in all three groups. It is concluded that the histochemical, biochemical and molecular mitochondrial changes in muscle are not specific for ALS, but accompany other NAs as well.