Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria

The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories—a regimen that increases life span by 30–40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.     

Network distribution of mitochondria and lipid droplets in human muscle fibres

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.     

High-performance liquid chromatography-based methods of enzymatic analysis: electron transport chain activity in mitochondria from human skeletal muscle

This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as approximately 10 mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40 microg/ml alamethicin and the reaction is terminated by H(2)SO(4), which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1-20 microM. NADH is quantitatively analyzed with a detection limit of 3-5 pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.     

Volume density and distribution of mitochondria in harbor seal (<Emphasis Type="Italic">Phoca vitulina</Emphasis>) skeletal muscle

Recent studies have shown that harbor seals (Phoca vitulina) have an increased skeletal muscle mitochondrial volume density that may be an adaptation for maintaining aerobic metabolism during diving. However, these studies were based on single samples taken from locomotory muscles. In this study, we took multiple samples from a transverse section of the epaxial (primary locomotory) muscles and single samples from the m. pectoralis (secondary locomotory) muscle of five wild harbor seals. Average mitochondrial volume density of the epaxial muscles was 5.6%, which was 36.6% higher than predicted for a terrestrial mammal of similar mass, and most (82.1%) of the mitochondria were interfibrillar, unlike athletic terrestrial mammals. In the epaxial muscles, the total mitochondrial volume density was significantly greater in samples collected from the deep (6.0%) compared with superficial (5.0%) regions. Volume density of mitochondria in the pectoralis muscle was similar (5.2%) to that of the epaxial muscles. Taken together, these adaptations reduce the intracellular distance between mitochondria and oxymyoglobin and increase the mitochondrial diffusion surface area. This, in combination with elevated myoglobin concentrations, potentially increases the rate of oxygen diffusion into mitochondria and prevents diffusion limitation so that aerobic metabolism can be maintained under low oxygen partial pressure that develops during diving.     

Influence of environmental temperature and energy intake on skeletal muscle respiratory enzymes and morphology

Influence of a cold (10 degrees C) or warm (35 degrees C) environment and a high or low level of energy intake on respiratory enzyme activities has been investigated in porcine skeletal muscle. Scanning microdensitometry was used to measure the reaction products from mitochondrial enzymes in individual slow- and fast-twitch muscle fibres. A cold environment was found to increase the activity of succinate dehydrogenase in both types of muscle fibre (P less than 0.001 for dark fibres, P less than 0.01 for light fibres) from young growing animals. Enzyme activity was also increased in animals on a low compared with a high energy intake (P less than 0.01) when living at 10 degrees C but not at 35 degrees C. Similar findings were obtained for NADH diaphorase and cytochrome oxidase aa3. The numbers of slow-twitch muscle fibres also increased after exposure to cold (P less than 0.01) and as a result of a low energy intake (P less than 0.01). These results are similar to those obtained in other species after exercise or as a result of peripheral arterial insufficiency. The extent to which they could be related to local tissue hypoxia or to changes in metabolic hormones is discussed.     

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O₂ consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.     

Identification of helical polyribosomes in sections of mature skeletal muscle fibers

Summary The localization and configurations of ribosomes in mature white skeletal muscle fibers of the rat were investigated. Differential visualization of ribosomes and glycogen granules was obtained by fixation with glutaraldehyde only and staining of the sections with uranyl acetate. Ribosomes are then electron dense and glycogen granules electron transparent. Their identity was ascertained by selective extractions of ribonucleic acid and polysaccharide.The vast majority of the ribosomes is not membrane-bound. They are located intermyofibrillarly (predominantly at the level of the I-bands), beneath the sarcolemma, and in the paranuclear cones of sarcoplasm. Occasionally short stretches of granular reticulum occur, often as characteristic double walled vesicles with ribosomes on the inner membrane only.Three main types of polysomal configurations are observed: rosettes of 4 to 6 ribosomes, helical arrays, and whorls of up to about 25 probably membrane-bound ribosomes. The average number of ribosomes in the extended helical configurations is estimated to be about 60. It is argued that these helices represent the polysomes instrumental in the synthesis of the large subunits of myosin. It is emphasized that helical polyribosomes are by no means typical of striated muscle, but probably represent a common configuration of large free polysomes.With the technical assistance of Tineke J. Hoogenboezem.     

Protein and collagen content of rat skeletal muscle following space flight

Summary Biochemical determinations of non-collagenous protein and hydroxyproline were made on rat skeletal muscles following 7 days of space flight aboard the NASA space shuttle mission SL-3. Relative to ground-based controls, the wet weight of each experimental muscle was significantly reduced. This was concomitant with a reduction in noncollagenous protein in the muscles. Protein concentration, however, was reduced only in slow-twitch muscles. The effect of space flight on the concentration and hydroxyproline content was different among the muscles. As a result, the loss of muscle mass in some muscles was the consequence of a reduction in both collagenous and non-collagenous proteins, while in others it was primarily the result of a non-collagenous protein loss.     

Activity of antioxidant enzymes in rat skeletal muscle and brown fat: effect of cold and propranolol

1. Effects of acute (6 h) and chronic (21 day) cold (6°C) exposure, as well as propranolol (15 mg/kg) on the activities of CuZnSOD, MnSOD and catalase in the rat skeletal muscle (SM) and brown adipose tissue (BAT), which are important sites of cold-induced thermogenesis, were investigated.

2. The changes in the activity of antioxidant enzymes were tissue specific and dependent on the duration of cold exposure. Thus, in the SM of acutely cold exposed rats, the activity of all antioxidant enzymes studied was elevated, whereas in the BAT the activity of both SOD_s decreased and that of catalase remained unchanged. In cold acclimated rats, the activity of all the three enzymes was increased in the BAT whereas in the SM, CuZnSOD activity was enhanced, MnSOD activity decreased and catalase activity returned to the control level.

3. Propranolol also differently altered the antioxidant enzyme activity in SM and BAT, alterations being dependent on the acclimation temperature. Thus, in room acclimated rats propranolol decreased the activity of all antioxidant enzymes in SM but did not affect those in BAT. However, in the SM propranolol prevented the elevation of MnSOD and catalase activities, induced by acute cold. In cold acclimated rats propranolol inhibited CuZnSOD activity in both SM and BAT but increased that of MnSOD.

Change of gene expression on protein uptake composition and hindlimb-suspension in rat skeletal muscle

[Purpose]

This study was to investigate changes in BCAT and BCKDH genes by Hindlimb-Suspension (HS) and protein intake composition (casein, Whey protein) in rats.

[Methods]

Following 5-day preliminary feeding, forty-eight male 5 weeks old Sprague Dawley albino rats (110g) divided into 17% protein intake group (24 rats) and 30% protein intake group (24 rats), and each group divided further into Hindlimb-Suspension group (HS; 12 rats) and control group(CON; 12 rats). Eventually, this study was performed with Whey protein intake group (HS; 6 rats, CON; 6 rats) and casein intake group (HS; 6 rats, CON; 6 rats). For analysis purposes, total RNA was extracted from isolated skeletal muscles, and mRNA expression was analyzed using Real Time PCR. Two-way ANOVA was performed to examine the difference in BCATm and BCKDH mRNA expression on protein uptake and myoatrophy. post-hoc test was perform on interaction if any, and significance level was set at p<0.05.

[Results]

In this study, BCATm and BCKDH gene analysis in rat skeletal muscles by hindlimb-suspension and protein intake composition resulted in significant higher BCATm expression in 30% dietary protein group and hindlimb-suspension group that control group. In addition, regarding BCKDH, BCKDH was significantly higher in hindlimb-suspended 30% protein intake group than control group.

[Conclusion]

Overall, protein intake and myoatrophy demonstrated close relationship in skeletal muscles. Therefore, it is likely to affect effectively in prevention or recovery of exercise induced muscle disorder. This effect is considered to be applied to maintain and improve health of not only athletes but also the general public. Additionally it would be applied in convalescent rehabilitation due to skeletal muscle atrophy.     

Functional argument for the existence of an avian nitric oxide synthase in muscle mitochondria: effect of cold acclimation

We report the first evidence of a mitochondrial NO synthase (mtNOS) in bird skeletal muscle. In vitro, mtNOS activity stimulated by l-arginine reduced intermyofibrillar mitochondrial oxygen uptake and ATP synthesis rates, stimulated endogenous H₂O₂ generation, but had no effect on oxidative phosphorylation efficiency. Arginine-induced effects were fully reversed by l-NAME, a known NOS inhibitor. When ducklings were cold exposed for 4 weeks, muscle mitochondria displayed an increased state 3 respiration, a reduced H₂O₂ generation but no significant alteration in mtNOS activity. We conclude that mtNOS is expressed in avian skeletal muscle.     

Effect of leucine uptake on hepatic and skeletal muscle gene expression in rats: a microarray analysis

[Purpose]

This study was performed to explore the physiological functions of leucine by exploring genes with leucine-dependent variability using DNA microarray.

[Methods]

Sprague-Dawley rats (n = 20) were separated into a HPD (30% High Protein Diet, n = 10) group and a NPD (0% Non Protein Diet, n = 10) group and fed a protein diet for 2 weeks. At the end of the 2-week period, the rats were fasted for 12-16 hours, further separated into subgroups within the HPD (Saline, n = 5, Leucine, n = 5) and NPD (Saline, n = 5, Leucine, n = 5) groups and administered with a leucine solution. The liver and muscles were harvested after 2 hours for RNA extraction. RNA purification from the isolated muscles and target gene identification using DNA chip were performed. The target gene was determined based on the results of the DNA chip experiment, and mRNA expression of the target gene was analyzed using Real-Time PCR.

[Results]

In the skeletal muscle, 27 genes were upregulated while 52 genes were down regulated after leucine administration in the NPD group. In the liver, 160 genes were up-regulated while 126 were down-regulated. The per2 gene was one of the genes with leucine-dependent induction in muscles and liver.

[Conclusion]

This study was performed to explore the physiological functions of leucine, however, a large number of genes showed variability. Therefore, it was difficult to definitively identify the genes linked with a particular physiological function. Various nutritional effects of leucine were observed. High variability in cytokines, receptors, and various membrane proteins were observed, which suggests that leucine functions as more than a nutrient. The interpretation may depend on investigators’ perspectives, therefore, discussion with relevant experts and the BCAA (Branched-Chain Amino Acids) society may be needed for effective utilization of this data.     

Isoforms of troponin during regeneration of chicken skeletal muscle fibers after cold injury

Summary The regeneration of skeletal muscle fibers of the adult chicken was examined after a focal injury brought about with a liquid-nitrogen cooled brass rod. Immunofluorescence microscopy with antibodies specific for troponin (TN) components (T, I, and C) from adult chicken breast and ventricular muscles showed the presence of different fiber types in both the anterior and posterior latissimus dorsi muscles. New fibers produced in the regions adjacent to the site of injury in both muscles exhibited the same immunoreactivities as those previously seen in embryonic skeletal muscles. As differentiation proceeded, regenerating cells lost their embryonic antigenicities and recovered their characteristic adult reactivities. These results indicate that, during regeneration from cold injury, skeletal muscles apparently pass again through an embryonic stage during which they synthesize embryonic-like TN isoforms.     

Early postnatal growth of skeletal muscle blood vessels of the rat

The development of blood vessels during the first three postnatal weeks was studied in the ventral stripe of the spinotrapezius muscle of the rat by use of India ink-gelatine injections, and electron microscopy. The number of terminal arterioles and collecting venules remained unchanged postnatally in the observed area. A remarkable proximodistal gradient of vascular development was apparent: while the basic structure of the hilar vessels remained unchanged in the time studied, the intramuscular arteries and veins matured gradually. More peripherally, gradual maturation of terminal and precapillary arterioles was observed. The capillary endothelium and the pericytes showed immature features, and remained unchanged during the time studied. An intense rebuilding activity was found in the endothelial cells of the growing venules, expressed by various forms of gaps, covered by an intact basal lamina and pericytes. Numerous mast cells and macrophages were found along all vessels. Intramuscular lymphatics were not present prior to the first postnatal week.     

The anticancer agent doxorubicin disrupts mitochondrial energy metabolism and redox balance in skeletal muscle

The combined loss of muscle strength and constant fatigue are disabling symptoms for cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and premature fatigue along with an increase in reactive oxygen species (ROS). As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin could negatively affect mitochondria by inhibiting respiratory capacity, leading to an increase in H₂O₂-emitting potential. Here we demonstrate a biphasic response of skeletal muscle mitochondria to a single doxorubicin injection (20 mg/kg). Initially at 2 h doxorubicin inhibits both complex I- and II-supported respiration and increases H₂O₂ emission, both of which are partially restored after 24 h. The relationship between oxygen consumption and membrane potential (ΔΨ) is shifted to the right at 24 h, indicating elevated reducing pressure within the electron transport system (ETS). Respiratory capacity is further decreased at a later time point (72 h) along with H₂O₂-emitting potential and an increased sensitivity to mitochondrial permeability transition pore (mPTP) opening. These novel findings suggest a role for skeletal muscle mitochondria as a potential underlying cause of doxorubicin-induced muscle dysfunction.     

Muscle metabolism and growth in Antarctic fishes (suborder Notothenioidei): evolution in a cold environment

The radiation of notothenioid fishes (order Perciformes) in the Southern Ocean provides a model system for investigating evolution and adaptation to a low temperature environment. The Notothenioid fishes comprising eight families, 43 genera and 122 species dominate the fish fauna in Antarctica. The diversification of the clade probably began 15–20 million years ago after the formation of the Antarctic Polar Front. The radiation was, therefore, associated with climatic cooling down to the present day temperature of −1.86 °C. Origins and Evolution of the Antarctic Biota Geological Society Special Publication No. 47, Geological Society of London. pp. 253–268). The success of the group has been closely linked with the evolution of glycopeptide and peptide antifreezes, which are amongst the most abundant proteins in blood and interstitial fluid. The radiation of the clade has been associated with disaptation (evolutionary loss of function) and recovery. For example, it is thought that the icefishes (Channichyidae) lost haemoglobin through a single mutational event leading to the deletion of the entire β-globin gene and the 5′ end of the linked α-globin gene, resulting in compensatory adaptations of the cardiovascular system. Phylogenetically based statistical methods also indicate a progressive and dramatic reduction in the number of skeletal muscle fibres (FN_max) at the end of the recruitment phase of growth in basal compared to derived families. The reduction in FN_max is associated with a compensatory increase in the maximum fibre diameter, which can reach 100 μm in slow and 600 μm in fast muscle fibres. At −1 to 0 °C, the oxygen consumption of isolated mitochondria per mg mitochondrial protein shows no evidence of up-regulation relative to mitochondria from temperate and tropical Perciform fishes. The mitochondria content of slow muscle fibres in Antarctic notothenioids is towards the upper end of the range reported for teleosts with similar lifestyles, reaching 50% in Channichthyids. High mitochondrial densities facilitate ATP production and oxygen diffusion through the membrane lipid compartment of the fibre. Modelling studies suggest that adequate oxygen flux in the large diameter muscle fibres of notothenioids is possible because of the reduced metabolic demand and enhanced solubility of oxygen associated with low temperature. At the whole animal level size-corrected resting metabolic rate fits on the same temperature relationship as for Perciformes from warmer climates. It seems likely that the additional energetic costs associated with antifreeze synthesis and high mitochondrial densities are compensated for by reductions in other energy requiring processes: a hypothesis that could be tested with detailed energy budget studies. One plausible candidate is a reduction in membrane leak pathways linked to the loss of muscle fibres, which would serve to minimise the cost of maintaining ionic gradients.     

Expression of nestin,desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating “spindle fibers”, 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.     

Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56 U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6 µg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79–84%) transfected muscle fibers with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle.     

Age-dependent regulation of skeletal muscle mitochondria by the thrombospondin-1 receptor CD47

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O₂ ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1-CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.