Home About us Contact
|
Home About us Contact | ||
|
|
||
Rat Skeletal Muscle (rat + skeletal_muscle)
Selected AbstractsDifferences in Local Environment Determine the Site of Physiological Angiogenesis in Rat Skeletal MuscleEXPERIMENTAL PHYSIOLOGY, Issue 5 2003I. Badr The specificity in location of angiogenesis to either glycolytic or oxidative fibre types, or muscle regions, was examined in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of rat. Angiogenesis was induced by mechanical means either with (chronic muscle stimulation) or without (muscle stretch by overload) changes in blood flow, treatments which invoked only minor changes in fibre type and fibre size. Proliferation estimated by PCNA labelling of cells co-localised with capillaries was very rare in control muscles, where it occurred mainly in the glycolytic regions, but was increased in both models of angiogenesis. However, when labelled capillaries were scored according to the type of surrounding fibres, only muscle stimulation significantly accentuated proliferation of capillaries surrounded by glycolytic fibres. We conclude that while mechanical stimuli are important for proliferation in glycolytic regions in both models, capillary growth occurs specifically around glycolytic fibres in that region when the angiogenic stimulus includes increased blood flow and/or increased metabolic demand. [source] Substrate Channelling in a Creatine Kinase System of Rat Skeletal Muscle Under Various pH ConditionsEXPERIMENTAL PHYSIOLOGY, Issue 1 2003M. Gregor The aim of this study was to evaluate myofibrillar creatine kinase (CK) activity and to quantify the substrate channelling of ATP between CK and myosin ATPase under different pH conditions within the integrity of myofibrils. A pure myofibrillar fraction was prepared using differential centrifugation. The homogeneity of the preparation and the purity of the fraction were confirmed microscopically and by enzymatic assays for contaminant enzyme activities. The specific activity of myofibrillar CK reached 584 ± 33 nmol PCr min,1 mg,1 at pH 6.75. Two methods were used to detect CK activity: (1) measurement of direct ATP production, and (2) measurement of PCr consumption. This method of evaluation has been tested in experiments with isolated creatine kinase. No discrepancy in CK activity between the methods was observed in the pH range tested (6.0-7.5). However, the same procedures resulted in a significant discrepancy between the amounts of reacted PCr and produced ATP within the pure myofibrillar fraction. This discrepancy represents the portion of ATP produced by the CK reaction, which is preferentially channelled to the myosin ATPase before diffusing into the bulk solution. The maximum evaluated difference reached 42.3 % at pH 6.95. The substrate channelling between myofibrillar-bound CK and myosin ATPase was evaluated under various pH levels within the physiological range and it reached a maximum value in a slightly acidic environment. These results suggest that ATP/ADP flux control by the CK system is more important at lower pH, corresponding to the physiological state of muscle fatigue. [source] Ethanol Feeding Impairs Insulin-Stimulated Glucose Uptake in Isolated Rat Skeletal Muscle: Role of Gs , and cAMPALCOHOLISM, Issue 8 2005Qiang Wan Background: The mechanism by which chronic alcohol consumption impairs insulin sensitivity is unclear. We investigated the role of the Gs ,,mediated pathway in decreasing insulin sensitivity in skeletal muscle after ethanol consumption. Methods: Sixty male Wistar rats, divided into four groups, received either distilled water (controls; group I) or ethanol, which was administered by a gastric tube as a single daily dose of 5 g/kg (group II), 2.5 g/kg (group III), or 0.5 g/kg (group IV). After 20 weeks, fasting plasma glucose and serum insulin levels were measured. The hyperinsulinemic-euglycemic clamp study was performed under anesthesia to estimate whole-body insulin sensitivity. Insulin-stimulated glucose uptake was measured in vitro in dissected gastrocnemius muscle. Expression of glut4, Gs ,, and Gi , was quantified using real-time PCR analysis and western blotting. cAMP levels were measured by ELISA. Results: Compared with controls, the following observations were made: (1) the hyperinsulinemic-euglycemic clamp study revealed impaired insulin action at the whole-body level after ethanol treatment; (2) chronic ethanol feeding at 5 g/kg and 2.5 g/kg significantly decreased both basal and insulin-stimulated glucose uptakes in isolated skeletal muscle (p < 0.05), which was accompanied by decreased expression of glut4 (p < 0.05); (3) Gs , (mRNA and protein) expression in skeletal muscle was significantly increased in all three ethanol groups (p < 0.05), and cAMP levels were also increased by ethanol treatment (p < 0.05); and (4) there was no significant change in Gi , expression in all three ethanol groups. Conclusions: Chronic ethanol exposure decreased insulin-induced glucose uptake in rat skeletal muscle, which was associated with increased expression of Gs ,. Because Gs , is a negative regulator of insulin sensitivity, the alteration in Gs , expression may contribute to the ethanol-induced impairment of insulin signal transduction. [source] Selective Long-Term Electrical Stimulation of Fast Glycolytic Fibres Increases Capillary Supply but not Oxidative Enzyme Activity in Rat Skeletal MusclesEXPERIMENTAL PHYSIOLOGY, Issue 5 2000S. Egginton Glycolytic fibres in rat extensor digitorum longus (EDL) and tibialis anterior (TA) were selectively activated, as demonstrated by glycogen depletion, by indirect electrical stimulation via electrodes implanted in the vicinity of the peroneal nerve using high frequency (40 Hz) trains (250 ms at 1 Hz) and low voltage (threshold of palpable contractions). This regime was applied 10 times per day, each bout being of 15 min duration with 60 min recovery, for 2 weeks. Cryostat sections of muscles were stained for alkaline phosphatase to depict capillaries, succinate dehydrogenase (SDH) to demonstrate oxidative fibres, and periodic acid-Schiff reagent (PAS) to verify glycogen depletion. Specific activity of hexokinase (HK), 6-phosphofructokinase, pyruvate kinase, glycogen phosphorylase and cytochrome c oxidase (COX) were estimated separately in homogenates of the EDL and the predominantly glycolytic cortex and oxidative core of the TA. Stimulation increased the activity of HK but not that of oxidative enzymes in fast muscles. Comparison of changes in oxidative capacity and capillary supply showed a dissociation in the predominantly glycolytic TA cortex. Here, COX was 3.9 ± 0.68 ,M min-1 (g wet wt)-1 in stimulated muscles compared with 3.7 ± 0.52 ,M min-1 (g wet wt)-1 in contralateral muscles (difference not significant), while the percentage of oxidative fibres (those positively stained for SDH) was also similar in stimulated (14.0 ± 2.8%) and contralateral (12.2 ± 1.9%) muscles. In contrast, the capillary to fibre ratio was significantly increased (2.01 ± 0.12 vs. 1.55 ± 0.04, P < 0.01). We conclude that capillary supply can be increased independently of oxidative capacity, possibly due to haemodynamic factors, and serves metabolite removal to a greater extent than substrate delivery. [source] Special gears for full-time engines: association of dystrophin,glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscleACTA PHYSIOLOGICA, Issue 4 2009Ugo Carraro No abstract is available for this article. [source] Correlation of dystrophin,glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscleACTA PHYSIOLOGICA, Issue 4 2009S. Masuda Abstract Aim:, The dystrophin,glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures in muscle fibres that link the intracellular cytoskeleton to the extracellular matrix. DGC and FAC proteins are abundant in slow-type muscles, indicating the structural reinforcement which play a pivotal role in continuous force output to maintain posture for long periods. The aim of the present study was to examine the expression of these structures across fast-type muscles containing different myosin heavy chain (MHC) isoform patterns which reflect the fatigue-resistant characteristics of skeletal muscle. Methods:, We measured the expression of dystrophin and ,1 integrin (representative proteins of DGC and FAC respectively) in plantaris, extensor digitorum longus, tibialis anterior, red and white portions of gastrocnemius, superficial portion of vastus lateralis and diaphragm, in comparison with soleus (SOL) and cardiac muscle from rats. Results:, The expression of dystrophin and ,1 integrin correlated positively with the percentage of type I, IIa and IIx MHC isoforms and negatively with that of type IIb MHC isoform in fast-type skeletal muscles, and their expression was abundant in SOL and cardiac muscle. Conclusion:, Our results support the idea that DGC and FAC are among the factors that explain the fatigue-resistant property not only of slow-type but also of fast-type skeletal muscles. [source] Metabolic cost of lengthening, isometric and shortening contractions in maximally stimulated rat skeletal muscleACTA PHYSIOLOGICA, Issue 2 2004J. G. M. Beltman Abstract Aim:, The present study investigated the energy cost of lengthening, isometric and shortening contractions in rat muscle (n = 19). Methods:, With electrical stimulation the rat medial gastrocnemius muscle was maximally stimulated to perform 10 lengthening, isometric and shortening contractions (velocity 25 mm s,1) under experimental conditions (e.g. temperature, movement velocity) that resemble conditions in human movement. Results:, Mean ± SD force,time-integral of the first contraction was significantly different between the three protocols, 2.4 ± 0.2, 1.7 ± 0.2 and 1.0 ± 0.2 N s, respectively (P < 0.05). High-energy phosphate consumption was not significantly different between the three modes of exercise but a trend could be observed from lengthening (7.7 ± 2.7 ,mol , P muscle,1) to isometric (8.9 ± 2.2 ,mol , P muscle,1) to shortening contractions (10.4 ± 1.6 ,mol , P muscle,1). The ratio of high-energy phosphate consumption to force,time-integral was significantly lower for lengthening [0.3 ± 0.1 ,mol , P (N s),1] and isometric [0.6 ± 0.2 ,mol , P (N s),1] contractions compared with shortening [1.2 ± 0.2 ,mol , P (N s),1] contractions (P < 0.05). Conclusion:, The present results of maximally stimulated muscles are comparable with data in the literature for voluntary human exercise showing that the energy cost of force production during lengthening exercise is ,30% of that in shortening exercise. The present study suggests that this finding in humans probably does reflect intrinsic muscle properties rather than effects of differential recruitment and/or coactivation. [source] Synthesis and degradation of type IV collagen in rat skeletal muscle during immobilization in shortened and lengthened positionsACTA PHYSIOLOGICA, Issue 4 2003A. M. Ahtikoski Abstract Aim:, Type IV collagen is a major protein in basement membranes surrounding and supporting skeletal muscle cells. In the present study, we tested the hypotheses that immobilization down-regulates synthesis and up-regulates degradation of type IV collagen in skeletal muscle. Methods:, mRNA level and concentration of type IV collagen as well as mRNA levels and activities of proteins involved in its degradation were analysed from soleus (SOL), gastrocnemius (GAS) and extensor digitorum longus muscles after immobilization in shortened and lengthened positions for 1, 3 and 7 days. Results:, Following immobilization, type IV collagen mRNA level was decreased in SOL and GAS suggesting down-regulated synthesis of this protein. The mRNA level and activity of matrix metalloproteinase-2 (proMMP-2) were increased in all muscles, while the activity of tissue inhibitor of metalloproteinase-2 was decreased in SOL and GAS. These findings reflect an increased capacity for degradation of type IV collagen. Conclusions: As a consequence of decreased synthesis/degradation ratio immobilization reduced the concentration of type IV collagen in all muscles. The regulation of type IV collagen through synthesis and/or degradation seems, however, to be muscle specific. Immobilization in lengthened position seems to delay and partly decrease the net degradation of type IV collagen. [source] Neutrophil,independent protective effect of r,metHuG,CSF in ischaemia,reperfusion injury in rat skeletal muscleINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 1 2000Article first published online: 15 JUN 200 [source] Differential activation of stress-responsive signalling proteins associated with altered loading in a rat skeletal muscleJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Inho Choi Abstract Skeletal muscle undergoes a significant reduction in tension upon unloading. To explore intracellular signalling mechanisms underlying this phenomenon, we investigated twitch tension, the ratio of actin/myosin filaments, and activities of key signalling molecules in rat soleus muscle during a 3-week hindlimb suspension and 2-week reloading. Twitch tension and myofilament ratio (actin/myosin) gradually decreased during unloading but progressively recovered to initial levels during reloading. To study the involvement of stress-responsive signalling proteins during these changes, the activities of protein kinase C alpha (PKC,) and three mitogen-activated protein kinases (MAPKs),c-Jun NH2 -terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 MAPK,were examined using immunoblotting and immune complex kinase assays. PKC, phosphorylation correlated positively with the tension (Pearson's r,=,0.97, P,<,0.001) and the myofilament ratio (r,=,0.83, P,<,0.01) over the entire unloading and reloading period. Treatment of the soleus muscle with a PKC activator resulted in a similar paralleled increment in both PKC, phosphorylation and the ,-sarcomeric actin expression. The three MAPKs differed in the pattern of activation in that JNK activity peaked only for the first hours of reloading, whereas ERK and p38 MAPK activities remained elevated during reloading. These results suggest that PKC, may play a pivotal role in converting loading stress to intracellular changes in contractile proteins that determine muscle tension. Differential activation of MAPKs may also help alleviate muscle damage, modulate energy transport and/or regulate the expression of contractile proteins upon altered loading. J. Cell. Biochem. © 2005 Wiley-Liss, Inc. [source] Melatonin inhibits the expression of the inducible isoform of nitric oxide synthase and nuclear factor kappa B activation in rat skeletal muscleJOURNAL OF PINEAL RESEARCH, Issue 1 2006María Alonso Abstract:, This study investigated whether the induction of inducible nitric oxide synthase (iNOS) produced by acute exercise in rat skeletal muscle could be prevented by melatonin and whether iNOS down-regulation was related to inhibition of nuclear factor kappaB (NF- ,B) activation. Male Wistar rats received melatonin i.p. at a dose of 1.0 mg/kg body weight 30 min before being exercised for 60 min on a treadmill at a speed of 25 m/min and a 10% slope. Exercise caused a significant induction of iNOS protein levels and a marked activation of NF- ,B that were significantly prevented in rats treated with melatonin. Exercise also resulted in increased I,B kinase, (IKK,) and phosphorylated I,B, protein levels, whereas I,B, content decreased. These effects were blocked by melatonin administration. The increase in the muscle concentration of thiobarbituric acid reactive substances and in the oxidized/reduced glutathione ratio induced by exercise was partially prevented by melatonin. Our data indicate that melatonin has potent protective effects against damage caused by acute exercise in rat muscle, preventing oxidative stress, NF- ,B activation and iNOS over-expression. These findings support the view that melatonin treatment, by abolishing the IKK/NF- ,B signal transduction pathway, might block the production of noxious mediators involved in the inflammatory process. [source] Ethanol Feeding Impairs Insulin-Stimulated Glucose Uptake in Isolated Rat Skeletal Muscle: Role of Gs , and cAMPALCOHOLISM, Issue 8 2005Qiang Wan Background: The mechanism by which chronic alcohol consumption impairs insulin sensitivity is unclear. We investigated the role of the Gs ,,mediated pathway in decreasing insulin sensitivity in skeletal muscle after ethanol consumption. Methods: Sixty male Wistar rats, divided into four groups, received either distilled water (controls; group I) or ethanol, which was administered by a gastric tube as a single daily dose of 5 g/kg (group II), 2.5 g/kg (group III), or 0.5 g/kg (group IV). After 20 weeks, fasting plasma glucose and serum insulin levels were measured. The hyperinsulinemic-euglycemic clamp study was performed under anesthesia to estimate whole-body insulin sensitivity. Insulin-stimulated glucose uptake was measured in vitro in dissected gastrocnemius muscle. Expression of glut4, Gs ,, and Gi , was quantified using real-time PCR analysis and western blotting. cAMP levels were measured by ELISA. Results: Compared with controls, the following observations were made: (1) the hyperinsulinemic-euglycemic clamp study revealed impaired insulin action at the whole-body level after ethanol treatment; (2) chronic ethanol feeding at 5 g/kg and 2.5 g/kg significantly decreased both basal and insulin-stimulated glucose uptakes in isolated skeletal muscle (p < 0.05), which was accompanied by decreased expression of glut4 (p < 0.05); (3) Gs , (mRNA and protein) expression in skeletal muscle was significantly increased in all three ethanol groups (p < 0.05), and cAMP levels were also increased by ethanol treatment (p < 0.05); and (4) there was no significant change in Gi , expression in all three ethanol groups. Conclusions: Chronic ethanol exposure decreased insulin-induced glucose uptake in rat skeletal muscle, which was associated with increased expression of Gs ,. Because Gs , is a negative regulator of insulin sensitivity, the alteration in Gs , expression may contribute to the ethanol-induced impairment of insulin signal transduction. [source] A Mutation in Mitochondrial Complex I Increases Ethanol Sensitivity in Caenorhabditis elegansALCOHOLISM, Issue 4 2003Ernst-Bernhard Kayser Background: The gene gas-1 encodes the 49-kDa subunit of complex I of the mitochondrial electron transport chain in Caenorhabditis elegans. A mutation in gas-1 profoundly increases sensitivity to ethanol and decreases complex I-dependent metabolism in mitochondria. Methods: Mitochondria were isolated from wild-type and gas-1 strains of C. elegans. The effects of ethanol on complex I-, II-, and III-dependent oxidative phosphorylation were measured for mitochondria from each strain. Reversibility of the effects of ethanol was determined by measuring oxidative phosphorylation after removal of mitochondria from 1.5 M ethanol. The effects of ethanol on mitochondrial structure were visualized with electron microscopy. Results: We found that ethanol inhibited complex I,, II,, and III,dependent oxidative phosphorylation in isolated wild-type mitochondria at concentrations that immobilize intact worms. It is important to note that the inhibitory effects of ethanol on mitochondria from either C. elegans or rat skeletal muscle were reversible even at molar concentrations. Complex I activity was lower in mitochondria from gas-1 animals than in mitochondria from wild-type animals at equal ethanol concentrations. Complex II activity was higher in gas-1 than in wild-type mitochondria at all concentrations of ethanol. No difference was seen between the strains in the sensitivity of complex III to ethanol. Conclusions: The difference in ethanol sensitivities between gas-1 and wild-type nematodes results solely from altered complex I function. At the respective concentrations of ethanol that immobilize whole animals, mitochondria from each strain of worms displayed identical rates of complex I-dependent state 3 respiration. We conclude that a threshold value of complex I activity controls the transition from mobility to immobility of C. elegans. [source] Clenbuterol increases muscle fiber size and GATA-2 protein in rat skeletal muscle in uteroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2008Diane Downie Abstract Certain ,2 -adrenoceptor agonists, such as clenbuterol, are known to elicit a muscle-specific anabolism or hypertrophy in both normal and catabolic muscle in a wide variety of species. However, the underlying mechanism(s) of the ,2 -agonist-induced anabolism remains unclear. This study aimed to determine the effects of clenbuterol administration in utero on skeletal muscle and to examine the underlying molecular mechanisms. Pregnant rats were fed clenbuterol (2 mg/kg diet) from Day 4 of gestation (4 dg) until weanling and fetal samples were taken from 13.5, 15.5, 17.5, and 19.5 dg and from 1d neonatal pups. Muscles were analyzed for total DNA, RNA and protein and sections examined morphologically for changes in muscle development. Western and immunohistochemical analyses were performed to identify changes in known myogenic signaling proteins. Clenbuterol increased the size of both fast and slow fibers in utero which was associated with a decreased DNA:protein ratio (28%) and an increased RNA:DNA ratio (36%). Additionally, drug treatment in utero induced a decrease in the fast:slow fiber ratio (38%). These myogenic changes were correlated with an increase in the GATA-2 hypertrophic transcription factor at both 17.5 dg (by 250%) and 19.5 dg (by 40%) in fetuses from clenbuterol treated dams. In addition, drug treatment resulted in increased membrane association of PKC-µ at 17.5 dg (325%) and increased PKC-, cytosolic abundance (40%) and PKC-, membrane abundance at 19.5 dg (250%). These results are the first demonstration that ,2 -agonists such as clenbuterol may act through upregulating the GATA-2 transcription factor and implicate certain PKC isoforms in the drug-induced regulation of skeletal muscle development. Mol. Reprod. Dev. 75: 785,794, 2008. © 2007 Wiley-Liss, Inc. [source] Endotoxemia does not limit energy supply in exercising rat skeletal muscleMUSCLE AND NERVE, Issue 4 2008Benoit Giannesini PhD Abstract Although depletion in high-energy phosphorylated compounds and mitochondrial impairment have been reported in septic skeletal muscle at rest, their impact on energy metabolism has not been documented during exercise. In this study we aimed to investigate strictly gastrocnemius muscle function non-invasively, using magnetic resonance techniques in endotoxemic rats. Endotoxemia was induced by injecting animals intraperitoneally at t0 and t0 + 24 h with Klebsiella pneumoniae lipopolysaccharides (at 3 mg kg,1). Investigations were performed at t0 + 48 h during a transcutaneous electrical stimulation protocol consisting of 5.7 min of repeated isometric contractions at a frequency of 3.3 HZ. Endotoxin treatment produced a depletion in basal phosphocreatine content and a pronounced reduction in oxidative adenosine triphosphate (ATP) synthesis capacity, whereas the resting ATP concentration remained unchanged. During the stimulation period, endotoxemia caused a decrease in force-generating capacity that was fully accounted for by the loss of muscle mass. It further induced an acceleration of glycolytic ATP production and an increased accumulation of adenosine diphosphate (ADP, an important mitochondrial regulator) that allowed a near-normal rate of oxidative ATP synthesis. Finally, endotoxemia did not affect the total rate of ATP production or the ATP cost of contraction throughout the whole stimulation period. These data demonstrate that, in an acute septic phase, metabolic alterations in resting muscle do not impact energy supply in exercising muscle, likely as a result of adaptive mechanisms. Muscle Nerve, 2008 [source] Inhibition of the initial wave of NF-,B activity in rat muscle reduces ischemia/reperfusion injuryMUSCLE AND NERVE, Issue 4 2001Sean T. Lille MD Abstract Nuclear factor kappaB (NF-,B) is thought to play an important role in the expression of genes expressed in response to ischemia/reperfusion (I/R) injury. In this report, the activation of NF-,B in rat skeletal muscle during reperfusion following a 4-h ischemic period was studied. NF-,B activation displayed a biphasic pattern, showing peak activities from 30 min to 3 h postperfusion and 6 h to 16 h postperfusion, with a decline to baseline binding activity levels between 3 h and 6 h. Inhibition of NF-,B activation was investigated using proline dithiocarbamate (Pro-DTC). NF-,B binding activity during reperfusion was significantly reduced by intravenous administration of Pro-DTC. Additionally, Pro-DTC resulted in decreased muscle edema and neutrophil activity, with an increased percentage of muscle survival compared with vehicle controls. These results demonstrate that NF-,B is activated during reperfusion in a biphasic manner and that the regulation of the initial phase of NF-,B activation affords physiological protection against a severe ischemic stress. Selective inhibition of NF-,B during early reperfusion may therefore be a therapeutic intervention for I/R injury. © 2001 John Wiley & Sons, Inc. Muscle Nerve 24: 534,541, 2001 [source] Compartmental relaxation and diffusion tensor imaging measurements in vivo in ,-carrageenan-induced edema in rat skeletal muscle,NMR IN BIOMEDICINE, Issue 6 2008Reuben H. Fan Abstract Integrated diffusion tensor T2 measurements were made on normal and edematous rat muscle, and the data were fitted with one- and two-compartment models, respectively. Edematous muscle exhibited a short-lived component (T2,=,28,±,6,ms), with diffusion characteristics similar to that of normal muscle, and a long-lived component (T2,=,96,±,27,ms), with greater mean apparent diffusion coefficient (ADC) and lower fractional anisotropy (FA). With this two-component description of diffusion and relaxation, values of ADC and FA estimated with a conventional pulsed-gradient spin-echo sequence will depend on the echo time, relative fraction of short-lived and long-lived water signals, and the intrinsic ADC and FA values within the tissue. On the basis of the relative differences in water diffusion properties between long-lived and short-lived water signals, as well as the similarities between the short-lived component and normal tissue, it is postulated that these two signal components largely reflect intracellular and extracellular water. Copyright © 2007 John Wiley & Sons, Ltd. [source] Proteomic DIGE analysis of the mitochondria-enriched fraction from aged rat skeletal musclePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2009Kathleen O'Connell Abstract Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria-enriched fraction from young adult versus aged muscle revealed an age-related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age-dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX-III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate-coenzyme A ligase, acyl-coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol-cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE-identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic-oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging. [source] High-precision isotopic analysis of palmitoylcarnitine by liquid chromatography/electrospray ionization ion-trap tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2006ZengKui Guo Single quadrupole gas chromatography/mass spectrometry (GC/MS) has been widely used for isotopic analysis in metabolic investigations using stable isotopes as tracers. However, its inherent shortcomings prohibit it from broader use, including low isotopic precision and the need for chemical derivatization of the analyte. In order to improve isotopic detection power, liquid chromatography/electrospray ionization ion-trap tandem mass spectrometry (LC/ESI-itMS2) has been evaluated for its isotopic precision and chemical sensitivity for the analysis of [13C]palmitoylcarnitine. Over the enrichment range of 0.4,10 MPE (molar % excess), the isotopic response of LC/ESI-itMS2 to [13C]palmitoylcarnitine was linear (r,=,1.00) and the average isotopic precision (standard deviation, SD) was 0.11 MPE with an average coefficient of variation (CV) of 5.6%. At the lower end of isotopic enrichments (0.4,0.9 MPE), the isotopic precision was 0.05 MPE (CV,=,8%). Routine analysis of rat skeletal muscle [13C4]palmitoylcarnitine demonstrated an isotopic precision of 0.03 MPE for gastrocnemius (n,=,16) and of 0.02 MPE for tibialis anterior (n,=,16). The high precision enabled the detection of a small (0.08 MPE) but significant (P,=,0.01) difference in [13C4]palmitoylcarnitine enrichments between the two muscles, 0.51 MPE (CV,=,5.8%) and 0.43 MPE (CV,=,4.6%), respectively. Therefore, the system demonstrated an isotopic lower detection limit (LDL) of ,0.1 MPE (2 × SD) that has been impossible previously with other organic mass spectrometry instruments. LC/ESI-itMS2 systems have the potential to advance metabolic investigations using stable isotopes to a new level by significantly increasing the isotopic solving power. Copyright © 2006 John Wiley & Sons, Ltd. [source] Regulation and function of Ca2+,calmodulin-dependent protein kinase II of fast-twitch rat skeletal muscleTHE JOURNAL OF PHYSIOLOGY, Issue 3 2007Adam J. Rose The activation and function of Ca2+,calmodulin-dependent kinase II (CaMKII) in contracting rat skeletal muscle was examined. The increase in autonomous activity and phosphorylation at Thr287 of CaMKII of gastrocnemius muscle in response to contractions in situ was rapid and transient, peaking at 1,3 min, but reversed after 30 min of contractions. There was a positive correlation between CaMKII phosphorylation at Thr287 and autonomous CaMKII activity. In contrast to the rapid and transient increase in autonomous CaMKII activity, the phosphorylation of the putative CaMKII substrate trisk95/triadin was rapid and sustained during contractions. There were no changes in CaMKII activity and phosphorylation or trisk95 phosphorylation in the resting contralateral muscles during stimulation. When fast-twitch muscles were contracted ex vivo, CaMKII inhibition resulted in a greater magnitude of fatigue as well as blunted CaMKII and trisk95 phosphorylation, identifying trisk95 as a physiological CaMKII substrate. In summary, skeletal muscle CaMKII activation was rapid and sustained during exercise/contraction and is mediated by factors within the contracting muscle, probably through allosteric activation via Ca2+,CaM. CaMKII may signal through trisk95 to modulate Ca2+ release in fast-twitch rat skeletal muscle during exercise/contraction. [source] AMP kinase activation with AICAR further increases fatty acid oxidation and blunts triacylglycerol hydrolysis in contracting rat soleus muscleTHE JOURNAL OF PHYSIOLOGY, Issue 2 2005Angela C. Smith Muscle contraction increases glucose uptake and fatty acid (FA) metabolism in isolated rat skeletal muscle, due at least in part to an increase in AMP-activated kinase activity (AMPK). However, the extent to which AMPK plays a role in the regulation of substrate utilization during contraction is not fully understood. We examined the acute effects of 5-aminoimidazole-4-carboxamide riboside (AICAR; 2 mm), a pharmacological activator of AMPK, on FA metabolism and glucose oxidation during high intensity tetanic contraction in isolated rat soleus muscle strips. Muscle strips were exposed to two different FA concentrations (low fatty acid, LFA, 0.2 mm; high fatty acid, HFA, 1 mm) to examine the role that FA availability may play in both exogenous and endogenous FA metabolism with contraction and AICAR. Synergistic increases in AMPK ,2 activity (+45%; P < 0.05) were observed after 30 min of contraction with AICAR, which further increased exogenous FA oxidation (LFA: +71%, P < 0.05; HFA: +46%, P < 0.05) regardless of FA availability. While there were no changes in triacylglycerol (TAG) esterification, AICAR did increase the ratio of FA partitioned to oxidation relative to TAG esterification (LFA: +65%, P < 0.05). AICAR significantly blunted endogenous TAG hydrolysis (LFA: ,294%, P < 0.001; HFA: ,117%, P < 0.05), but had no effect on endogenous oxidation rates, suggesting a better matching between TAG hydrolysis and subsequent oxidative needs of the muscle. There was no effect of AICAR on the already elevated rates of glucose oxidation during contraction. These results suggest that FA metabolism is very sensitive to AMPK ,2 stimulation during contraction. [source] Reversible changes in Ca2+ -activation properties of rat skeletal muscle exposed to elevated physiological temperaturesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2002Chris van der Poel Exposure of relaxed rat extensor digitorum longus (EDL; predominantly fast-twitch) muscle to temperatures in the upper physiological range for mammalian skeletal muscle (43-46 °C) led to reversible alterations of the contractile activation properties. These properties were studied using the mechanically skinned fibre preparation activated in Ca2+ -buffered solutions. The maximum Ca2+ -activated force (maximum force per cross-sectional area) and the steepness of force-pCa (-log10[Ca2+]) curves as measured by the Hill coefficient (nH) reversibly decreased by factors of 8 and 2.5, respectively, when the EDL muscle was treated at 43 °C for 30 min and 5 and 2.8, respectively, with treatment at 46 °C for 5 min. Treatment at 47 °C for 5 min produced an even more marked depression in maximum specific force, which fully recovered after treatment, and in the Hill coefficient, which did not recover after treatment. After all temperature treatments there was no change in the level of [Ca2+] at which 50 % maximum force was generated. The temperature-induced depression in force production and steepness of the force-pCa curves were shown to be associated with superoxide (O2,) production in muscle (apparent rate of O2, production at room temperature, 0.055 ± 0.008 nmol min,1 (g wet weight),1; and following treatment to 46 °C for 5 min, 1.8 ± 0.2 nmol min,1 (g wet weight),1) because 20 mm Tiron, a membrane-permeant O2, scavenger, was able to markedly suppress the net rate of O2, production and prevent any temperature-induced depression of contractile parameters. The temperature-induced depression in force production of the contractile apparatus could be reversed either by allowing the intact muscle to recover for 3-4 h at room temperature or by treatment of the skinned fibre preparation with dithiothreitol (a potent reducing agent) in the relaxing solution. These results demonstrate that mammalian skeletal muscle has the ability to uncouple force production reversibly from the activator Ca2+ as the temperature increases in the upper physiological range through an increase in O2, production. [source] OCTN2 is associated with carnitine transport capacity of rat skeletal musclesACTA PHYSIOLOGICA, Issue 1 2010Y. Furuichi Abstract Aim:, Carnitine plays an essential role in fat oxidation in skeletal muscles; therefore carnitine influx could be crucial for muscle metabolism. OCTN2, a sodium-dependent solute carrier, is assumed to transport carnitine into various organs. However, OCTN2 protein expression and the functional importance of carnitine transport for muscle metabolism have not been studied. We tested the hypothesis that OCTN2 is expressed at higher levels in oxidative muscles than in other muscles, and that the carnitine uptake capacity of skeletal muscles depends on the amount of OCTN2. Methods:, Rat hindlimb muscles (soleus, plantaris, and the surface and deep portions of gastrocnemius) were used for Western blotting to detect OCTN2. Tissue carnitine uptake was examined by an integration plot analysis using l -[3H]carnitine as a tracer. Tissue carnitine content was determined by enzymatic cycling methods. The percentage of type I fibres was determined by histochemical analysis. Results:, OCTN2 was detected in all skeletal muscles although the amount was lower than that in the kidney. OCTN2 expression was significantly higher in soleus than in the other skeletal muscles. The amount of OCTN2 was positively correlated with the percentage of type I fibres in hindlimb muscles. The integration plot analysis revealed a positive correlation between the uptake clearance of l -[3H]carnitine and the amount of OCTN2 in skeletal muscles. However, the carnitine content in soleus was lower than that in other skeletal muscles. Conclusion:, OCTN2 is functionally expressed in skeletal muscles and is involved in the import of carnitine for fatty acid oxidation, especially in highly oxidative muscles. [source] Caffeine and theophylline block insulin-stimulated glucose uptake and PKB phosphorylation in rat skeletal musclesACTA PHYSIOLOGICA, Issue 1 2010A. J. Kolnes Abstract Aim:, Caffeine and theophylline inhibit phosphatidylinositol 3-kinase (PI3-kinase) activity and insulin-stimulated protein kinase B (PKB) phosphorylation. Insulin-stimulated glucose uptake involves PI3-kinase/PKB, and the aim of the present study was to test the hypothesis that caffeine and theophylline inhibit insulin-stimulated glucose uptake in skeletal muscles. Methods:, Rat epitrochlearis muscles and soleus strips were incubated with insulin and different concentrations of caffeine and theophylline for measurement of glucose uptake, force development and PKB phosphorylation. The effect of caffeine was also investigated in muscles stimulated electrically. Results:, Caffeine and theophylline completely blocked insulin-stimulated glucose uptake in both soleus and epitrochlearis muscles at 10 mm. Furthermore, insulin-stimulated PKB Ser473 and Thr308 and GSK-3, Ser9 phosphorylation were blocked by caffeine and theophylline. Caffeine reduced and theophylline blocked insulin-stimulated glycogen synthase activation. Caffeine stimulates Ca2+ release and force development increased rapidly to 10,20% of maximal tetanic contraction. Dantrolene (25 ,m), a well-known inhibitor of Ca2+ -release, prevented caffeine-induced force development, but caffeine inhibited insulin-stimulated glucose uptake in the presence of dantrolene. Contraction, like insulin, stimulates glucose uptake via translocation of glucose transporter-4 (GLUT4). Caffeine and theophylline reduced contraction-stimulated glucose uptake by about 50%, whereas contraction-stimulated glycogen breakdown was normal. Conclusion:, Caffeine and theophylline block insulin-stimulated glucose uptake independently of Ca2+ release, and the likely mechanism is via blockade of insulin-stimulated PI3-kinase/PKB activation. Caffeine and theophylline also reduced contraction-stimulated glucose uptake, which occurs independently of PI3-kinase/PKB, and we hypothesize that caffeine and theophylline also inhibit glucose uptake in skeletal muscles via an additional and hitherto unknown molecule involved in GLUT4 translocation. [source] Different adaptations of alpha-actinin isoforms to exercise training in rat skeletal musclesACTA PHYSIOLOGICA, Issue 3 2009Y. Ogura Abstract Aim:, Alpha (,)-actinins are located in the skeletal muscle Z-line and form actin,actin cross-links. Mammalian skeletal muscle has two isoforms: ,-actinin-2 and ,-actinin-3. However, the response of ,-actinin to exercise training is little understood. Therefore, the current study examined the effects of exercise training on the expression level of two ,-actinin isoforms in skeletal muscles. Methods:, Twelve male Wistar rats were assigned randomly to a control (C; n = 6) or exercise training (T; n = 6) group. After T animals were trained on an animal treadmill for 9 weeks, ,-actinin-2 and ,-actinin-3 levels in the plantaris, white and red gastrocnemius muscles were analysed. In addition, changes in the myosin heavy chain (MyHC) composition were assessed, and muscle bioenergetic enzyme activities were measured. Results:, Results show that exercise training increased ,-actinin-2 expression levels in all muscles (P < 0.05). However, no significant difference was found in ,-actinin-3 expression levels between C and T animals. Subsequent MyHC analyses of all muscle showed an MyHC shift with direction from IIb to IIa. Furthermore, enzymatic analysis revealed that exercise training improved enzyme activities related to aerobic metabolism. Conclusion:, The results of this study demonstrate that exercise training alters the expression level of ,-actinin at the isoform level. Moreover, the increase in expression levels of ,-actinin-2 is apparently related to alteration of skeletal muscle: its aerobic capacity is improved. [source] The expression patterns of Pax7 in satellite cells during overload-induced rat adult skeletal muscle hypertrophyACTA PHYSIOLOGICA, Issue 4 2009M. Ishido Abstract Aim:, Activated satellite cells (SCs) have the ability to reacquire a quiescent, undifferentiated state. Pax7 plays a crucial role in allowing activated SCs to undergo self-renewal. Because the increase in the SC population is induced during overload-induced skeletal muscle hypertrophy, it is possible that Pax7-regulated SC self-renewal is involved in the modulation of the SC population during the functional overload of skeletal muscles. However, the characteristics of the expression patterns of Pax7 in SCs during the functional overload of adult skeletal muscles are poorly understood. Methods:, Using immunohistochemical approaches, we examined the temporal and spatial expression patterns of Pax7 expressed in SCs during the functional overloading of rat skeletal muscles. Results:, The time course of Pax7 expression in SCs was similar to that of the expression of the differentiation regulatory factor myogenin during the early stage of functional overload. However, the percentage of SCs that expressed Pax7 was markedly higher than that of the SCs that expressed myogenin. Coexpression of Pax7 and myogenin was not detected in SCs. In addition, the expression of cyclin-dependent kinase inhibitor p21, which regulates cell cycle arrest and differentiation, was not detected in Pax7-positive SCs. Conclusion:, These results suggest that Pax7-regulated self-renewal of SCs may be induced during the early stage of functional overload and may contribute to modulating the SC population in hypertrophied muscles. Furthermore, it was suggested that the numbers of SCs which underwent self-renewal may be higher than that of SCs which were provided as the additional myonuclei for hypertrophying myofibres. [source] Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activityJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2005M. Zimowska Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N -acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity. © 2005 Wiley-Liss, Inc. [source] Endurance training adaptations modulate the redox,force relationship of rat isolated slow-twitch skeletal musclesCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 1-2 2003David R Plant Summary 1.,Studies have shown that, in isolated skeletal muscles, maximum isometric force production (Po) is dependent on muscle redox state. Endurance training increases the anti-oxidant capacity of skeletal muscles, a factor that could impact on the force-producing capacity following exogenous exposure to an oxidant. We tested the hypothesis that 12 weeks treadmill training would increase anti-oxidant capacity in rat skeletal muscles and alter their response to exogenous oxidant exposure. 2.,At the conclusion of the 12 week endurance-training programme, soleus (slow-twitch) muscles from trained rats had greater citrate synthase (CS) and catalase (CAT) activity compared with soleus muscles from untrained rats (P < 0.05). In contrast, CAT activity of extensor digitorum longus (EDL; fast-twitch) muscles from trained rats was not different to EDL muscles of untrained rats. The CS activity was lower in EDL muscles from trained compared with untrained rats (P < 0.05). 3.,Equilibration with exogenous hydrogen peroxide (H2O2, 5 mmol/L) increased the Po of soleus muscles from untrained rats for the duration of treatment (30 min), whereas the Po of EDL muscles was affected biphasically, with a small increase initially (after 5 min), followed by a more marked decrease in Po (after 30 min). The H2O2 -induced increase in Po of soleus muscles from trained rats was less than that in untrained rats (P < 0.05), but no differences were observed in the Po of EDL muscles following training. 4.,The results indicate that 12 weeks endurance running training conferred adaptations in soleus but not EDL muscles. These adaptations were associated with an attenuation of the oxidant-induced increase in Po of soleus muscles from trained compared with untrained rats. We conclude that endurance training-adapted soleus muscles have a slightly altered redox,force relationship. [source] | ||||||||||||||||||||||||||||