Regulation of VEGF and bFGF mRNA expression and other proliferative compounds in skeletal muscle cells

The role of muscle contraction, prostanoids, nitric oxide and adenosine in the regulation of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endothelial cell proliferative compounds in skeletal muscle cell cultures was examined. VEGF and bFGF mRNA, protein release as well as the proliferative effect of extracellular medium was determined in non-stimulated and electro-stimulated rat and human skeletal muscle cells. In rat skeletal muscle cells these aspects were also determined after treatment with inhibitors and/or donors of nitric oxide (NO), prostanoids and adenosine. Electro-stimulation caused an elevation in the VEGF and bFGF mRNA levels of rat muscle cells by 33% and 43% (P < 0.05), respectively, and in human muscle cells VEGF mRNA was elevated by 24%. Medium from electro-stimulated human, but not rat muscle cells induced a 126% higher (P < 0.05) endothelial cell proliferation than medium from non-stimulated cells. Cyclooxygenase inhibition of rat muscle cells induced a 172% increase (P < 0.05) in VEGF mRNA and a 104% increase in the basal VEGF release. Treatment with the NO donor SNAP (0.5 M) decreased (P < 0.05) VEGF and bFGF mRNA by 42 and 38%, respectively. Medium from SNAP treated muscle cells induced a 45% lower (P < 0.05) proliferation of endothelial cells than control medium. Adenosine enhanced the basal VEGF release from muscle cells by 75% compared to control. The present data demonstrate that contractile activity, NO, adenosine and products of cyclooxygenase regulate the expression of VEGF and bFGF mRNA in skeletal muscle cells and that contractile activity and NO regulate endothelial cell proliferative compounds in muscle extracellular fluid.     

Dilinoleoyl-phosphatidic acid mediates reduced IRS-1 tyrosine phosphorylation in rat skeletal muscle cells and mouse muscle

Aims/hypothesis Insulin resistance in skeletal muscle is strongly associated with lipid oversupply, but the intracellular metabolites and underlying mechanisms are unclear. We therefore sought to identify the lipid intermediates through which the common unsaturated fatty acid linoleate causes defects in IRS-1 signalling in L6 myotubes and mouse skeletal muscle. Materials and methods Cells were pre-treated with 1 mmol/l linoleate for 24 h. Subsequent insulin-stimulated IRS-1 tyrosine phosphorylation and its association with the p85 subunit of phosphatidylinositol 3-kinase were determined by immunoblotting. Intracellular lipid species and protein kinase C activation were modulated by overexpression of diacylglycerol kinase ɛ, which preferentially converts unsaturated diacylglycerol into phosphatidic acid, or by inhibition of lysophosphatidic acid acyl transferase with lisofylline, which reduces phosphatidic acid synthesis. Phosphatidic acid species in linoleate-treated cells or muscle from insulin-resistant mice fed a safflower oil-based high-fat diet that was rich in linoleate were analysed by mass spectrometry. Results Linoleate pretreatment reduced IRS-1 tyrosine phosphorylation and p85 association. Overexpression of diacylglycerol kinase ɛ reversed the activation of protein kinase C isoforms by linoleate, but paradoxically further diminished IRS-1 tyrosine phosphorylation. Conversely, lisofylline treatment restored IRS-1 phosphorylation. Mass spectrometry indicated that the dilinoleoyl-phosphatidic acid content increased from undetectable levels to almost 20% of total phosphatidic acid in L6 cells and to 8% of total in the muscle of mice fed a high-fat diet. Micelles containing dilinoleoyl-phosphatidic acid specifically inhibited IRS-1 tyrosine phosphorylation and glycogen synthesis in L6 cells. Conclusions/interpretation These data indicate that linoleate-derived phosphatidic acid is a novel lipid species that contributes independently of protein kinase C to IRS-1 signalling defects in muscle cells in response to lipid oversupply. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorised users.     

Defects of fatty acid oxidation in skeletal muscle

Fatty acids are important substrates for resting and exercising skeletal muscle. Defects of fatty acid oxidation result in muscle pain and weakness, and there is often lipid accumulation in muscle fibres. Only a few of the several possible enzyme defects have been found and some of these have responded to therapy. Some techniques for the investigation of fatty acid oxidation are outlined.     

Melatonin prevents oxidative stress-mediated mitochondrial permeability transition and death in skeletal muscle cells

Abstract:  Oxidative stress-induced mitochondrial dysfunction plays a crucial role in the pathogenesis of a wide range of diseases including muscle disorders . In this study, we demonstrate that melatonin readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptosis of primary muscle cultures prepared from C57BL/6J mice. In particular, melatonin (10⁻⁴–10⁻⁶  m ) fully prevented myotube death induced by tert -butylhydroperoxide ( t -BHP; 10 μ m –24 hr) as assessed by acid phosphatase, caspase-3 activities and cellular morphological changes. Using fluorescence imaging, we showed that the mitochondrial protection provided by melatonin was associated with an inhibition of t -BHP-induced reactive oxygen species generation. In line with this observation, melatonin prevented t -BHP-induced mitochondrial depolarization and mitochondrial permeability transition pore (PTP) opening. This was associated with a highly reduced environment as reflected by an increased glutathione content and an increased ability to maintain mitochondrial pyridine nucleotides and glutathione in a reduced state. Using isolated mitochondria, in a similar manner as cyclosporin A, melatonin (10⁻⁸–10⁻⁶  m ) desensitized the PTP to Ca²⁺ and prevented t -BHP-induced mitochondrial swelling, pyridine nucleotide and glutathione oxidation. In conclusion, our findings suggest that inhibition of the PTP essentially contributes to the protective effect of melatonin against oxidative stress in myotubes.     

Human skeletal muscle satellite cells: aging,oxidative stress and the mitotic clock

Normal satellite cell cultures, isolated from human skeletal muscle, have a limited proliferative capacity and inevitably reach replicative senescence. In this study, we have focused on the consequences of a single oxidative stress by hydrogen peroxide (H(2)O(2)) on both proliferative capacity and myogenic characteristics. Treatment with 1mM H(2)O(2) for 30 min causes a small decrease in the viability and lifespan while the number of cells which are able to proliferate, decreases dramatically. This premature arrest of the cells in a non-proliferative state was not due to spontaneous differentiation since there was no increase in the number of myogenin positive cells. This stress did not affect the myogenicity of the cells or their ability to differentiate and fuse to form multinucleated myotubes. In addition, the mitotic clock does not seem to be modified by oxidative stress treatment since the rate of telomere shortening was similar in H(2)O(2)-treated and control cells. This could be the consequence of the high level of oxygen consumption with an even higher level of ROS being produced in skeletal muscle than in other tissues which would be counteracted by an increase in the antioxidant defense system.     

吸烟减少大鼠骨骼肌细胞胰岛素受体基因表达

应用RT-PCR及免疫组化技术分别检测实验大鼠骨骼肌细胞胰岛素受体基因mRNA及蛋白表达.结果显示正常吸烟组、高脂饲养吸烟组及糖尿病吸烟组大鼠骨骼肌细胞胰岛索受体基因mRNA的表达显著低于各自对照组(0.50±0.06对0.84±0.09,0.38±0.01对0.59±0.05,0.37±0.05对0.55±0.05,均P<0.01),蛋白含量亦明显低于各自对照组(6.99±0.53对8.89±0.36,5.17±0.29对7.53±0.53,2.16±0.56对5.03±0.79,均P<0.01),这可能是长期吸烟导致胰岛素抵抗的分子机制之一.     

Diffusivity of myoglobin in intact skeletal muscle cells.

We report a method that allows us to determine the diffusion coefficient of native myoglobin in intact and mechanically unaffected red muscle fibers. The method is based on an optical recording of intracellular diffusion of metmyoglobin, which is produced inside the cells by photooxidation of oxymyoglobin with a UV light pulse. We find a myoglobin diffusivity of 1.2 x 10(-7) cm2/s (22 degrees C), which is only 1/10th of the value measured in very dilute myoglobin solutions and 1/5th of the value obtained from measurements in solutions of myoglobin at 18 g/dl. The latter value often has been used in model calculations of oxygen transport to tissue incorporating myoglobin-facilitated oxygen diffusion. Recalculating facilitated diffusion with the value obtained by us implies that its contribution to total intracellular oxygen transport is of minor importance. Furthermore, it shows that sterical hindrance to myoglobin diffusion is dominated by the muscle-cell architecture rather than by the overall protein concentration of the muscle fiber.     

Specific phosphorylation at serine-283 of alpha tropomyosin from frog skeletal and rabbit skeletal and cardiac muscle.

Tropomyosin, extracted from the leg muscle of frogs that had been injected with [32P]orthophosphate, was fractionated into two components, alpha and beta, on a CM-cellulose column. Radioactivity was associated only with the alpha component. A single phosphorylation site was located at serine-283 (pentultimate at the COOH-terminal end) of the frog alpha tropomyosin. The same phosphorylated peptide was recovered in low yields from both rabbit skeletal alpha and cardiac tropomyosin. The presence of covalently bound phosphate in alpha tropomyosin and its absence in the beta component of rabbit skeletal muscle was suggested by 31P NMR spectroscopy. The amino acid sequences around the phosphorylation sites of frog and rabbit tropomyosin are identical. Because this sequence is not similar to any other known phosphorylation site in proteins, this indicates the existence of either specific kinase or phosphatase that can distinguish between alpha and beta tropomyosins. In a model proposed for the head-to-tail overlap of alpha tropomyosin molecules, one O-phosphoserine-283 residue could form a salt linkage with lysine-6 on one side of the overlap region and another with lysine-12 on the other side. This would predict a difference in the stability of polymers of phosphorylated and nonphosphorylated alphaalpha and alphabeta dimers of tropomyosin.     

Rostrocaudal gradient of transgene expression in adult skeletal muscle.

Transgenic mice were produced in which expression of the reporter gene chloramphenicol acetyltransferase (CAT) is controlled by regulatory elements of a rodent myosin light chain gene. CAT activity was readily detectable in muscles of these mice but negligible in a variety of nonmuscle tissues. Unexpectedly, levels of CAT expression varied greater than 100-fold from muscle to muscle, forming a gradient in which a muscle's position in the rostrocaudal axis was correlated with its level of CAT enzyme activity and abundance of CAT mRNA. Thus, rostral muscles (innervated by cranial nerves) had the lowest levels of CAT, thoracic muscles had intermediate levels, and caudal muscles (innervated through lumbar and sacral roots) had the highest levels. We established that myosin light chain sequences are responsible for the gradient of CAT expression but observed no strong gradient of endogenous myosin light chain expression. We argue that elements that are silent or masked by other sequences in their native context are revealed in the transgene and that the rostrocaudal gradient of gene expression they produce reveals the existence of a positionally graded endogenous regulator of gene expression. These transgenic mice provide evidence that cells in adult mammals retain "positional information" of a sort hitherto studied largely in embryos. The transgene they express may provide a means for determining how such positional values are generated and maintained.     

Age-related changes in the skeletal muscle cells

Skeletal muscle cells of young and old humans and CBA/Ca mice were examined electronmicroscopically. The mitochondria in old subjects and mice showed age-related changes. The cristae of mitochondria became irregularly spaced, disrupted and replaced by lamellar, myelin-like structures. Giant mitochondria were often visible. They contain lipofuscin in the myofibrils, too, which was often in close relationship with the damaged mitochondria. Comparing these mitochondrial changes with those demonstrated in the lymphocytes in authors' earlier reports, it can be stated that mitochondrial changes occur more frequently in the skeletal muscle cells than in the lymphocytes.     

Reduced exercise tolerance in CHF may be related to factors other than impaired skeletal muscle oxidative capacity

BACKGROUND: We sought to determine whether skeletal muscle oxidative capacity, fiber type proportions, and fiber size, capillary density or muscle mass might explain the impaired exercise tolerance in chronic heart failure (CHF). Previous studies are equivocal regarding the maladaptations that occur in the skeletal muscle of patients with CHF and their role in the observed exercise intolerance.Methods and results Total body O(2) uptake (VO(2peak)) was determined in 14 CHF patients and 8 healthy sedentary similar-age controls. Muscle samples were analyzed for mitochondrial adenosine triphosphate (ATP) production rate (MAPR), oxidative and glycolytic enzyme activity, fiber size and type, and capillary density. CHF patients demonstrated a lower VO(2peak) (15.1+/-1.1 versus 28.1+/-2.3 mL.kg(-1).min(-1), P<.001) and capillary to fiber ratio (1.09+/-0.05 versus 1.40+/-0.04; P<.001) when compared with controls. However, there was no difference in capillary density (capillaries per square millimeter) across any of the fiber types. Measurements of MAPR and oxidative enzyme activity suggested no difference in muscle oxidative capacity between the groups. CONCLUSIONS: Neither reductions in muscle oxidative capacity nor capillary density appear to be the cause of exercise limitation in this cohort of patients. Therefore, we hypothesize that the low VO(2peak) observed in CHF patients may be the result of fiber atrophy and possibly impaired activation of oxidative phosphorylation.     

Intense and exhaustive exercise induce oxidative stress in skeletal muscle

ObjectiveTo assess the oxidative stress and antioxidant defense system in the skeletal muscle of male albino rats subjected to strenuous exercise programme.MethodsWistar strain albino rats were subjected to exhaustive swimming exercise programme daily for a period of five days. The thiobarbituric acid reactive substances (TBARS), conjugated dienes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase were measured in the gastrocnemius muscle of the exercised animals.ResultsThe elevated levels of TBARS and conjugated dienes indicated the oxidative stress in the gastrocemius muscle of the exercised animals. The depleted activity levels of superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in the exercise animals indicated the increased oxidative stress and decreased antioxidative defense system in the muscle.ConclusionsThe study suggests that prolonged strenuous exercise programme can induce oxidative stress and therefore an optimal level of exercise schedule should be advocated to obtain the maximum benefit of exercise programme.     

Angiotensin II, oxidative stress and skeletal muscle wasting

Muscle atrophy (cachexia) is a muscle wasting syndrome associated with several pathological conditions in humans such as congestive heart failure, diabetes, AIDS, cancer and renal failure, and the presence of cachexia worsens outcome. Many of the conditions associated with cachexia are accompanied by stimulation of the renin-angiotensin system and elevation in angiotensin II (ang II) levels. Ang II infusion induces skeletal muscle atrophy in rodents and mechanisms include increased expression of the E3 ligases atrogin-1/MuRF-1, an elevated rate of ubiquitin-proteasome mediated proteolysis and increased reactive oxygen species (ROS) levels, closely mimicking conditions of human cachexia. Ang II-induced oxidative stress contributes to muscle atrophy in a mouse model. Nicotinamide adenine dinucleotide phosphate oxidase- and mitochondria-derived ROS contribute to ang II-induced oxidative stress. Specific targeting of ROS and nicotinamide adenine dinucleotide phosphate oxidase/mitochondria cross-talk could be a beneficial, novel therapy to treat cachexia.     

OLETF大鼠骨骼肌的氧化应激和Bcl-xL表达及罗格列酮干预的研究

目的探讨OLETF大鼠骨骼肌的氧化应激和BcbxL表达及罗格列酮（RGZ）的干预效应。方法设LETO大鼠为正常对照（NC）组,OI,ETF大鼠分为RGZ组和糖尿病（DM）组,测骨骼肌组织中一氧化氮（NO）、总超氧化物歧化酶（,r-S（）D）、谷胱甘肽（GSH）和丙二醛（MDA）,对骨骼肌细胞Bcl-xL表达情况进行检测。结果与NC组相比,DIM组骨骼肌组织中NO、T-SOD、GSH和骨骼肌细胞Bcl-xL阳性表达强度降低（P〈0．05或P〈0．01）,MDA升高（P〈0．01）;RGZ组相对于DM组NO、T-SOD和Bcl-XL阳性表达强度升高（P〈0．01）,MDA降低（P〈0．05）。结论T2DM本身可能通过促进骨骼肌组织氧化损伤加重细胞凋亡,而罗格列酮可能通过降低其氧化损伤缓解骨骼肌细胞凋亡,对骨骼肌有保护作用。