Cellular redox activity of coenzyme Q10: effect of CoQ10 supplementation on human skeletal muscle

In this paper, we report results obtained from a continuing clinical trial on the effect of coenzyme Q 10 (CoQ 10 ) administration on human vastus lateralis (quadriceps) skeletal muscle. Muscle samples, obtained from aged individuals receiving placebo or CoQ 10 supplementation (300 mg per day for four weeks prior to hip replacement surgery) were analysed for changes in gene and protein expression and in muscle fibre type composition. Microarray analysis (Affymetrix U95A human oligonucleotide array) using a change in gene expression of 1.8-fold or greater as a cutoff point, demonstrated that a total of 115 genes were differentially expressed in six subject comparisons. In the CoQ 10 -treated subjects, 47 genes were up-regulated and 68 down-regulated in comparison with placebo-treated subjects. Restriction fragment differential display analysis showed that over 600 fragments were differentially expressed using a 2.0-fold or greater change in expression as a cutoff point. Proteome analysis revealed that, of the high abundance muscle proteins detected (2086 ±115), the expression of 174 proteins was induced by CoQ 10 while 77 proteins were repressed by CoQ 10 supplementation. Muscle fibre types were also affected by CoQ 10 treatment; CoQ 10 -treated individuals showed a lower proportion of type I (slow twitch) fibres and a higher proportion of type IIb (fast twitch) fibres, compared to age-matched placebo-treated subjects. The data suggests that CoQ 10 treatment can act to influence the fibre type composition towards the fibre type profile generally found in younger individuals. Our results led us to the conclusion that coenzyme Q 10 is a gene regulator and consequently has wide-ranging effects on over-all tissue metabolism. We develop a comprehensive hypothesis that CoQ 10 plays a major role in the determination of membrane potential of many, if not all, sub-cellular membrane systems and that H 2 O 2 arising from the activities of CoQ 10 acts as a second messenger for the modulation of gene expression and cellular metabolism.     

Androgen receptor enhances myogenin expression and accelerates differentiation

Animal and clinical studies indicated that the androgen-AR signaling pathway is required for appropriate development of sexually dimorphic skeletal muscles and increases lean muscle mass, muscle strength, and muscle protein synthesis. However, the detailed mechanisms by which the androgen-AR signaling pathway regulates skeletal muscle development need further study at the molecular level. C2C12 myoblast cells stably transfected with the Flag-tagged AR were used to analyze the role of androgen-AR signaling pathway in skeletal muscle development. The results indicate that the androgen-AR signaling pathway may suppress skeletal myoblast cell growth and accelerate myoblast cell differentiation via enhanced myogenin expression. This is a first report showing the role of androgen-AR signaling pathway in regulation of myoblast cell growth and myogenic regulatory factors.     

Vascular endothelial growth factor is expressed in endothelial cells isolated from skeletal muscles of nitric oxide synthase knockout mice during prazosin-induced angiogenesis

In skeletal muscles, angiogenesis can be induced by increases in wall shear stress. To identify molecules involved in the angiogenic process, a method based on the use of BS-1 lectin-coated magnetic beads was developed to isolate a cellular fraction enriched in microvascular endothelial cells which are directly exposed to wall shear stress. Using such cellular fractions from skeletal muscles of C57 mice in which angiogenesis was induced by administration with the alpha(1)-adrenergic antagonist prazosin, we found the concentration of vascular endothelial growth factor (VEGF) increased in correlation to the duration of the prazosin stimulus. In contrast, the angiopoietin-2/tie-2 system was not changed even after 4days of prazosin treatment. In neuronal nitric oxide synthase (nNOS) knockout mice, the VEGF concentration was also elevated after prazosin treatment but remained almost unchanged in endothelial nitric oxide synthase (eNOS) knockout mice. However, eNOS (and not nNOS) knockout mice expressed higher levels of VEGF under non-stimulated conditions as compared to C57 mice. These results suggest that VEGF produced in endothelial cells is involved in angiogenesis in skeletal muscles of mice responding to the administration of systemic vasodilators. NO derived from eNOS and nNOS may be an important regulator of the angiogenic response in skeletal muscles in vivo.     

Interleukin-10 expression after intramuscular DNA electrotransfer: kinetic studies

A set of monoclonal antibodies that recognizes a Trypanosoma cruzi 45-kDa protein was produced and used to characterize this molecule and study its role in trypanosome adhesion to heart myoblasts. We found that the 45-kDa protein is a surface mucin, is expressed only in invasive trypomastigotes, but not in noninvasive epimastigotes or amastigotes, and is released by the trypanosome in culture medium. One of the monoclonal antibodies (Mab B5) from this set inhibits the attachment of trypomastigotes to heart myoblasts preventing trypanosome entry, whereas the others (Mabs B4 and F1) do not. This inhibition was seen with the B5 hybridoma culture supernatant, with the purified Mab B5 IgG or with Mab B5 Fab fragments. These novel findings identify the 45-kDa mucin as a new T. cruzi ligand that is used by invasive forms of this organism to adhere to heart myoblasts.     

A longitudinal study on hand and wrist skeletal maturation in chimpanzees ( Pan troglodytes), with emphasis on growth in linear dimensions

Skeletal maturation in the chimpanzee hand and wrist (the RUS system; radius, ulna, and short bones) was studied both longitudinally and cross-sectionally. Maturity states were evaluated in each of the 13 bones of the RUS system based on the TW2 method (Tanner and Whitehouse method), and the RUS score was calculated by the summation of scores for these bones. Individual variation was examined by means of residual curves and pseudo-velocity curves of RUS score and anterior trunk length (ATL). Norms of the age change pattern in RUS skeletal maturation and the growth of ATL were determined for each sex, and the relationships among ATL growth and skeletal and reproductive maturation were examined. We found a fairly good relationship between ATL growth and RUS skeletal maturation. Comparison of growth and development between humans and chimpanzees showed that growth characteristics are coupled with each other at puberty in male chimpanzees and in both sexes of humans. Although nutritional condition influenced ATL growth in infancy, it had no effect on the RUS maturational process. Social relationships appeared to influence both ATL growth and RUS maturation. Analyses on relationships between RUS skeletal maturation, ATL growth, and reproductive maturation, showed that RUS skeletal maturation is a good indicator of "physiological age".     

Models in which many cross-bridges attach simultaneously can explain the filament movement per ATP split during muscle contraction

Contractile filaments in skeletal muscle are moved by less than 2 nm for each ATP used. If just one cross-bridge is attached to each thin filament at any instant then this distance represents the fundamental myosin cross-bridge step size (i.e. the distance one cross-bridge moves a thin filament in one ATP-splitting cycle). However, most contraction models assume many cross-bridges are attached at any instant along each thin filament. The purpose of this study was to establish whether the net filament sliding per ATP used could be explained quantitatively in terms of a cross-bridge model in which multiple cross-bridges are attached along each thin filament. It was found that the relationship between net filament sliding per ATP split and the load against which the muscle shortens is compatible with such a model and furthermore predicts that the cross-bridge step size is between 7.5 and 12.5 nm over most of the range of loads. These values were similar for different muscle fibre types.     

Mitochondrial,sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats

Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1+/-0.1 to 3.6+/-0.14 nmol mg protein(-1), such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise.     

The Specific Force of Single Intact Extensor Digitorum Longus and Soleus Mouse Muscle Fibers Declines with Aging

In the present study we measured, for the first time, the isometric specific force (SF, force normalized to cross sectional area) generated by single intact fibers from fast- (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from young adult (2–6), middle-aged (12–14) and old (20–24 month-old) mice. SF has also been measured in single intact flexor digitorum brevis fibers from young mice. Muscle fibers have been classified into fast- or slow-twitch based on the contraction kinetics. Maximum SF recorded in EDL and soleus fibers from young and middle-aged mice did not differ significantly. A significant age-dependent decline in maximum SF was recorded in EDL and soleus fibers from young or middle-aged to old mice. The SF was 377 ± 18, 417 ± 20 and 279 ± 18 kPa for EDL fibers from young, middle-aged and old mice, respectively and 397 ± 17, 405 ± 24 and 320 ± 33 kPa for soleus fibers from age-matched mice, respectively. The frequency needed to elicit maximum force in EDL and soleus fibers from middle-aged to old mice did not differ significantly. In conclusion, the specific force developed by both fast and slow-twitch single intact muscle fibers declines with aging and more significantly in the former. Received: 14 July 2000/Revised: 7 September 2000     

Effects of ATP-sensitive Potassium Channel Regulators on Chloride Channels in the Sarcoplasmic Reticulum Vesicles from Rabbit Skeletal Muscle

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K⁺ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl⁻ channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K _i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I _max and average mean current, I′) and kinetic parameters (frequency of opening F _o , probability of the channel being open P _o and mean open time, T _o , of the SCl channel. Glibenclamide increased mean closed time, T _c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P _o , and T _o in the absence of ATP and Mg²⁺ but it had no effect on I _max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I _max, whereas cromakalim reduced P _o and I′ by increasing channel transitions to the closed state and reducing T _o without affecting I _max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] _cis or [glibenclamide] _cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] _cis and [glibenclamide] _cis highlights the therapeutic potential of these drugs in regulating the Ca²⁺-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998