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

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

Effect of long term starvation on the biochemical composition of the muscles and hepatopancreas in the crab Menippe rumphii (Fabricius) (Crustacea: Brachyura)

During the process of long term starvation both muscles and hepatopancreas are affected in their biochemical composition at different rates. During early days of starvation an increase in the muscular and hepatopancreatic glycogen is observed. At the same time a simultaneous decrease in the muscular lipid content is also observed. At a slightly later period a decrease in the hepatopancreatic lipid content is also noticed. This amount of decrease is slow in the early days of starvation and rapid in later days. Decrease in the muscular and hepatopancreatic protein content is observed when there is not an adequate quantity of hepatopancreatic lipid to be consumed.     

Myostatin shows a specific expression pattern in pig skeletal and extraocular muscles during pre- and post-natal growth

Abstract Myogenesis is driven by an extraordinary array of cellular signals that follow a common expression pattern among different animal phyla. Myostatin (mstn) is a secreted growth factor that plays a pivotal role in skeletal muscle mass regulation. The aim of the present study was to investigate mstn expression in a large mammal (the pig) in order to ascertain whether distinct expression changes of this factor might be linked to the fiber-type composition of the muscle examined and/or to specific developmental stages. To assess the expression pattern of mstn in relation to myogenic proliferative (Pax7 and MyoD) and differentiative (myogenin) markers, we evaluated muscles with different myosin heavy-chain compositions sampled during pre- and post-natal development and on myogenic cells isolated from the same muscles. Skeletal muscles showed higher levels of mRNA for mstn and all other genes examined during fetal development than after birth. The wide distribution of mstn was also confirmed by immunohistochemistry experiments supporting evidence for cytoplasmic staining in early fetal periods as well as the localization in type 1 fibers at the end of the gestation period. Extraocular muscles, in contrast, did not exhibit decreasing mRNA levels for mstn or other genes even in adult samples and expressed higher levels of both mstn mRNA and protein compared with skeletal muscles. Experiments carried out on myogenic cells showed that mstn mRNA levels decreased when myoblasts entered the differentiation program and that cells isolated at early post-natal stages maintained a high level of Pax7 expression. Our results showed that mstn had a specific expression pattern whose variations depended on the muscle type examined, thus supporting the hypothesis that at birth, porcine myogenic cells continue to be influenced by hyperplastic/proliferative mechanisms.     

Effects of long-term resistance exercise training on autophagy in rat skeletal muscle of chloroquine-induced sporadic inclusion body myositis

Purpose

We examined whether resistance exercise training restores impaired autophagy functions caused by Chloroquine (CQ)-induced Sporadic Inclusion Body Myositis (sIBM) in rat skeletal muscle.

Methods

Male wistar rats were randomly assigned into three groups: Sham (n = 6), CQ (n = 6), and CQ + Exercise (CE, n = 6). To create a rat model of sIBM, rats in the CQ and CE group were intraperitoneally injected with CQ 5 days a week for 16 weeks. Rats in the CE group performed resistance exercise training 3 times a week for 8 weeks in conjunction with CQ starting from week 9 to week 16. During the training period, maximal carrying load, body weight, muscle weight, and relative muscle weight were measured. Autophagy responses were examined by measuring specific markers.

Results

While maximal carrying capacity for resistance exercise training was dramatically increased in the CE group, no significant changes occurred in the skeletal muscle weight as well as in the relative muscle weight of CE compared to the other groups. CQ treatment caused significant increases in the levels of Beclin-1 and p62, and decreases in the levels of LAMP-2 proteins. Interestingly, no significant differences in the LC3-II/I ratio or the LC3-II protein levels were observed. Although CQ-treatment groups suppressed the levels of the potent autophagy inducer, BNIP3, p62 levels were decreased in only the CE group.

Conclusion

Our findings demonstrate that sIBM induced by CQ treatment results in muscle degeneration via impaired autophagy and that resistance exercise training improves movable loading activity. Finally, regular exercise training may provide protection against sIBM by enhancing the autophagy flux through p62 protein.     

Alteration of mitochondrial oxidative capacity during porcine preadipocyte differentiation and in response to leptin

Mitochondrial apparatus is a fundamental aspect in cell, serving for amino acid biosynthesis, fatty acid oxidation (FAO), and ATP production. In this article, we investigated the change of mitochondrial oxidative capacity during porcine adipocyte differentiation and in response to leptin. Rhodamine 123 staining analysis showed about 2-fold increase of mitochondrial membrane electric potential in differentiated adipocyte in comparison with preadipocyte. The mRNA expression of Cytochromes c (Cyt c), carnitine palmitoyltransferase 1 (CPT1), and malate dehydrogenases (MDH) increased markedly (P < 0.05), but that of UCP2 decreased (P < 0.05). Moreover PGC-1α and UCP3 was very low and showed no changes during the adipocyte differentiation. The protein expression of Cyt c and the enzyme activity of Cytochrome c oxidase (COX) increased with preadipocyte differentiation, but cellular ATP level decreased. Furthermore, at the level of 10 and 100 ng/ml leptin not only selectively increased the gene expression of PGC-1α, CPT1, Cyt c, UCP2, and UCP3 (P < 0.05), but also enhanced COX enzyme activity which related to mitochondrial FAO. There is no change of Mitochondrial membrane electric potential and ATP level in cell treated by leptin. These results suggested Mitochondrial is not only critical in FAO, but also play an important role in adipogenesis.     

骨骼肌急性钝挫伤修复过程中自噬相关因子的表达变化

目的：本研究通过观察SD大鼠骨骼肌急性钝挫伤修复过程中自噬相关因子的表达变化,探讨骨骼肌损伤修复可能的生物学机制。方法：30只SD雄性大鼠,随机选取6只作为对照组,其余24只用打击器打击后建立腓肠肌急性钝挫伤模型,然后随机分为4组（n=6）,各组分别在造模前及造模后3 d、5 d、7 d、14 d取材,HE染色观察损伤部位腓肠肌形态学变化,透射电镜观察损伤部位腓肠肌超微结构变化,Western blot检测腓肠肌自噬相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）、泛素结合蛋白P62表达水平,RT-PCR检测腓肠肌自噬相关基因（atg） atg7、atg10、atg12、atg16L1 mRNA表达水平。结果：HE染色显示：与对照组相比,骨骼肌损伤后5 d炎细胞浸润达到高峰,7 d可见明显的新生肌细胞,14 d损伤已初步愈合。电镜观察显示：与对照组相比,损伤后3 d,5 d,7 d线粒体肿胀明显、空泡化增多,Z线从消失到飘移增粗,肌质网扩张程度逐渐好转,14 d接近对照组水平。Western blot显示：骨骼肌损伤在自然恢复3 d、5 d、7 d、14 d过程中,LC3-Ⅱ与P62总体呈现先升高后降低的趋势,其中3 d、5 d、7 d组LC3-Ⅱ表达较对照组与14 d组明显升高（P<0.01）,同样损伤后第3天P62表达达到高峰（P<0.01）,14 d恢复至正常水平。RTPCR显示：骨骼肌损伤自然恢复3 d、5 d、7 d、14 d过程中,atg10 mRNA表达呈现先降低后升高的趋势,其中3 d、5 d、7 d组atg10 mRNA表达较对照组与14 d组明显降低（P<0.01）;atg7、atg12、atg16L1 mRNA表达总体呈现先升高后降低的趋势,其中3 d、5 d、7 d组表达较对照组与14 d组显著升高（P<0.01,P<0.05,P<0.01）。结论：骨骼肌急性钝挫伤后,自噬相关因子表达随损伤的修复而呈现规律性变化,提示自噬参与骨骼肌损伤的修复,推测骨骼肌急性钝挫伤的修复速度可能与细胞自噬水平有关。     

Chaotic dynamics of the surface potential of human skeletal muscles determined in electromyography

A new method for differential evaluation of electromyographic data on straited muscles of human lower extremities was developed. This method is based on nonlinear dynamics and thermodynamics and can be used for identification of pathologies. The distance between two trajectories of the potential of two symmetric muscles was the main measured characteristic of coordinated muscle work. These data were used to determine the Lyapunov exponent and the time of forgetting initial conditions, which reflect the generally chaotic dynamics of muscle activity. Application of the theory of deterministic chaos to analysis of electromyographic patterns can improve the diagnosis of peripheral nervous system diseases and the efficacy of treatment control. Quantitation of nonlinear dynamic parameters of muscle activity, clear data representation, high prognostic information content of the Lyapunov exponent and Kolmogorov entropy are among the advantages of the new method.     

Antioxidant effect of diphenyl diselenide on oxidative stress caused by acute physical exercise in skeletal muscle and lungs of mice

This study was designed to examine if diphenyl diselenide (PhSe)₂, an organoselenium compound, attenuates oxidative stress caused by acute physical exercise in skeletal muscle and lungs of mice. Swiss mice were pre‐treated with (PhSe)₂ (5 mg kg^‐1 day^‐1) for 7 days. At the 7th day, the animals were submitted to acute physical exercise which consisted of continuous swimming for 20 min. The animals were euthanized 1 and 24 h after the exercise test. The levels of thiobarbituric acid reactive species (TBARS), non‐protein thiols (NPSH) and ascorbic acid and the activity of catalase (CAT) were measured in the lungs and skeletal muscle of mice. Glycogen content was determined in the skeletal muscle of mice. Parameters in plasma (urea and creatinine) were determined. The results demonstrated an increase in TBARS levels induced by acute physical exercise in the skeletal muscle and lungs of mice. Animals submitted to exercise showed an increase in non‐enzymatic antioxidant defenses (NPSH and ascorbic acid) in the skeletal muscle. In lungs of mice, activity of CAT was increased. (PhSe)₂ protected against the increase in TBARS levels and ameliorated antioxidant defenses in the skeletal muscle and lungs of mice submitted to physical exercise. These results indicate that acute physical exercise caused a tissue‐specific oxidative stress in the skeletal muscle and lungs of mice. (PhSe)₂ protected against oxidative damage induced by acute physical exercise in mice. Copyright © 2009 John Wiley & Sons, Ltd.     

BmCalpains are involved in autophagy and apoptosis during metamorphosis and after starvation in Bombyx mori

Apoptosis and autophagy play crucial roles during Bombyx mori metamorphosis and in response to various adverse conditions, including starvation. Recently, calpain, one of the major intracellular proteases, has been reported to be involved in apoptosis and autophagy in mammals. BmATG5 and BmATG6 have been identified to mediate apoptosis following autophagy induced by 20‐hydroxyecdysone and starvation in B. mori. However, B. mori calpains and their functions remain unclear. In this study, phylogenetic analysis of calpains from B. mori, Drosophila melanogaster and Homo sapiens were performed and the results showed distinct close relationships of BmCalpain‐A/B with DmCalpain‐A/B, BmCalpain‐C with DmCalpain‐C, and BmCalpain‐7 with HsCalpain‐7. Then, the expression profiles of BmCalpains were analyzed by quantitative real‐time polymerase chain reaction, and results showed that expression of BmCalpain‐A/B, BmCalpain‐C and BmCalpain‐7 was significantly increased during B. mori metamorphosis and induced in the fat body and midgut of starved larvae, which is consistent with the expression profiles of BmAtg5, BmAtg6 and BmCaspase‐1. Moreover, the apoptosis‐associated cleavage of BmATG6 in Bm‐12 cells was significantly enhanced when BmCalpain‐A/B and BmCalpain‐7 were induced by starvation, and was partially inhibited by the inhibitor of either calpain or caspase, but completely inhibited when both types of inhibitors were applied together. Our results indicated that BmCalpains, including BmCalpain‐A/B, ‐C and ‐7, may be involved in autophagy and apoptosis during B. mori metamorphosis and after starvation, and may also contribute to the apoptosis‐associated cleavage of BmATG6.     

Ubiquitin expression is up-regulated in human and rat skeletal muscles during aging

In this study, we have used two-dimensional electrophoresis, protein sequencing, immunoblotting, and immunohistochemistry to identify proteins that were differentially expressed during aging in human and rat skeletal muscles. Ubiquitin was identified. It was expressed at high levels in old fast-twitch muscles but at low levels in young fast-twitch muscles. It was also discovered that exogenous ubiquitin could suppress the growth of C2C12 cells, in vitro. The reduction in C2C12 cell growth was not attributed to an increase in apoptosis but to an inhibition in cell cycle entry. Furthermore, it was possible to induce muscles to degenerate in vivo by injecting a high dose of exogenous ubiquitin into young healthy skeletal muscles. These results suggest that hyperactivity of the ubiquitin-proteasome pathway is involved in the aging process of fast-twitch muscles. In addition, ubiquitin-dependent growth suppression in satellite cells may be associated with the poor healing potential of old skeletal muscles.     

The skeletal muscles of rotifers and their innervation

The skeletal muscles of rotifers are monocellular or occasionally bicellular. They display great diversity of cytological features correlated to their functional differentiation. The cross-striated fibers of some retractors are fast contracting and relaxing, with A-band lengths of 0.7 µm to 1.6 µm, abundant sarcoplasmic reticulum and dyads. Other retractors and the circular muscles are tonic fibers (A band > 3 µm), stronger (large volume of myoplasm) or with greater endurance (superior volume of mitochondria/ myoplasm). All of these retractor muscles are coupled by gap junctions and are innervated at two symmetrical points; they constitute two motor units implicated in withdrawal behaviour.The muscles inserted on the ciliary roots of the cingulum control swimming. They are multi-innervated and each of them constitute one motor unit. They have characteristics of very fast fibers; the shortest A-band length is 0.5 µm in Asplanchna.All the skeletal muscles of bdelloids are smooth or obliquely striated as are some skeletal muscles of monogononts. These muscles are well suited for maximum shortening and are either phasic or tonic fibers.All rotifer skeletal muscles originate from ectoderm and contain thin and thick myofilaments whose diameters are identical to those of actin and myosin filaments in vertebrate fast muscles or in insect flight muscles. There are no paramyosinic features in the thick myofilaments. The insertion, innervation, coupling by gap junctions and other cytological differentiations of rotifer skeletal muscles are reviewed and their phylogeny discussed.     

Pitx3-CreER mice showing restricted Cre expression in developing ocular lens and skeletal muscles

To generate temporally controlled inactivation or activation of interested genes in Pitx3-expressing cells, the tamoxifen-inducible form of Cre, CreER(T2), was inserted into the Pitx3 locus of a mouse BAC clone. Following a single dose of tamoxifen, Cre activity in Pitx3-CreER(T2) transgenic mice was observed in the ocular lens and skeletal muscles but not in the central nervous system at various embryonic stages. This mouse line provides a reagent for driving inducible Cre-dependent recombination in the lens and skeletal muscles during embryonic development.     

Cryopreservation of mitochondria and mitochondrial function in cardiac and skeletal muscle fibers

Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze-thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze-thawing conditions, high rates of adenosine 5(')-diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5(')-triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze-thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.     

Quantitative evaluation of tryptophan oxidation in actin and troponin I from skeletal muscles using a rat model of acute oxidative stress

Increased levels of “ROS” cause oxidative stress and are believed to play a key role in the development of age‐related diseases and mammalian aging, e.g. through the oxidation of residues, at or close to, the protein surface. In this study, we have investigated the effects of ROS on tryptophan residues in alpha skeletal actin and troponin I (fast skeletal muscle isoform) using an established rat model of acute oxidative stress induced by X‐ray irradiation. In the control samples (no oxidative stress), the single Trp residue of troponin I (position 161) and the four tryptophan residues present in actin (positions 79, 86, 340, and 356) were only oxidized at very low levels. Post‐irradiation, the level of oxidized versions increased for most positions within 3 h. Tryptophan residues located inside the proteins, however, required longer time periods. Based on the increment masses of the tryptophan positions calculated from the b‐ and y‐ion series of the tandem mass spectra, the following oxidation products of tryptophan were detected: kynurenine; oxolactone; hydroxytryptophan or oxindolylalanine (isobaric); hydroxykynurenine; dioxindolylalanine, N‐formylkynurenine or dihydroxytryptophan (all three isobaric); and hydroxyl‐N‐formylkynurenine, with mass gains relative to tryptophan of 4, 14, 16, 20, 32, and 48 u, respectively. Despite a partial recovery after 24 h, the degree of oxidation of all oxidized versions was still higher than in the control samples.     

Age-related oxidative stress and antioxidant capacity in heat-stressed broilers

We aimed to evaluate the effects of acute heat stress (HS) and age on the redox state in broilers aged 21 and 42 days. We evaluated the expression of genes related to antioxidant capacity, the production of hydrogen peroxide (H₂O₂), and the activity of antioxidant enzymes in the liver, as well as oxidative stress markers in the liver and plasma. The experiment had a completely randomized factorial design with two thermal environments (thermoneutral and HS, 38°C for 24 h) and two ages (21 and 42 days). Twenty-one-day-old animals exposed to HS showed the highest thioredoxin reductase 1 (TrxR1) (P<0.0001) and glutathione synthetase (GSS) (P<0.0001) gene expression levels. Age influenced the expression of the thioredoxin (Trx) (P=0.0090), superoxide dismutase (SOD) (P=0.0194), glutathione reductase (GSR) (P<0.0001) and glutathione peroxidase 7 (GPx7) (P<0.0001) genes; we observed greater expression in birds at 21 days than at 42 days. Forty-two-day-old HS birds showed the highest H₂O₂ production (222.31 pmol dichlorofluorescein produced/min×mg mitochondrial protein). We also verified the effects of age and environment on the liver content of Glutathione (GSH) (P<0.0001 and P=0.0039, respectively) and catalase (CAT) enzyme activity (P=0.0007 and P=0.0004, respectively). Higher GSH content and lower CAT activity were observed in animals from the thermoneutral environment compared with the HS environment and in animals at 21 days compared with 42 days. Broilers at 42 days of age had higher plasma creatinine content (0.05 v. 0.01 mg/dl) and higher aspartate aminotransferase activity (546.50 v. 230.67 U/l) than chickens at 21 days of age. Our results suggest that under HS conditions, in which there is higher H₂O₂ production, 21-day-old broilers have greater antioxidant capacity than 42-day-old animals.