Differential effects of acute changes in cell Ca2+ concentration on myofibrillar and non-myofibrillar protein breakdown in the rat extensor digitorum longus muscle in vitro. Assessment by production of tyrosine and N tau-methylhistidine.
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ABSTRACT:
SUBMITTER: Goodman MN
PROVIDER: S-EPMC1147533 | biostudies-other | 1987 Jan
REPOSITORIES: biostudies-other
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Project description:The present study characterized total and myofibrillar protein breakdown rates in a muscle preparation frequently used in vitro, i.e. incubated extensor digitorum longus (EDL) and soleus (SOL) muscles of young rats. Total and myofibrillar protein breakdown rates were assessed by determining net production by the incubated muscles of tyrosine and 3-methylhistidine (3-MH) respectively. Both amino acids were determined by h.p.l.c. Both total and myofibrillar protein breakdown rates were higher in SOL than in EDL muscles and were decreased by incubating the muscles maintained at resting length, rather than flaccid. After fasting for 72 h, total protein breakdown (i.e. tyrosine release) was increased by 73% and 138% in EDL muscles incubated flaccid and at resting length respectively. Net production of tyrosine by SOL muscle was not significantly altered by fasting. In contrast, myofibrillar protein degradation (i.e. 3-MH release) was markedly increased by fasting in both muscles. When tissue was incubated in the presence of 1 munit of insulin/ml, total protein breakdown rate was inhibited by 17-20%, and the response to the hormone was similar in muscles incubated flaccid or at resting length. In contrast, myofibrillar protein breakdown rate was not altered by insulin in any of the muscle preparations. The results support the concepts of individual regulation of myofibrillar and non-myofibrillar proteins and of different effects of various conditions on protein breakdown in different types of skeletal muscle. Thus determination of both tyrosine and 3-MH production in red and white muscle is important for a more complete understanding of protein regulation in skeletal muscle.
| S-EPMC1131240 | biostudies-other
Project description:AbstractGanglion is the most common soft tissue mass in the foot and can be painful and affect comfort wearing shoes. The usual treatment of a ganglion is conservative: careful neglect, manual rupture, or aspiration. When the lesion is recurrent or painful, surgical excision is recommended. The purpose of this Technical Note is to describe the extraganglionic approach of endoscopic ganglionectomy of the extensor digitorum longus tendon. This surgery has the advantage of being minimally invasive and having better cosmetic result, with less surgical trauma to the soft tissue.Level of evidenceLevel 1: foot and ankle; Level 2: other (ganglion).
| S-EPMC8416964 | biostudies-literature
Project description:I. Experimental Design: a. Type of experiment: Time course b. Experimental factors: 3 month old mice subjected to lengthening contractions of the extensor digitorum longus muscle (EDL). Samples of EDL collected at 6 and 72 h and compared to non-treated control. c. How many hybridizations in exp: 9 d. Common reference used for all hyb: no e. Quality control steps: All arrays used from same lot. Triplicate hybridizations performed for each time point. II. Samples used, extracts, preparation and labeling: Animals. Nine male C57BL/6 mice (3-4 mo of age, 27.8 ± 3.3 g body mass, Harlan Sprague-Dawley, Indianapolis, IN). Muscle Injury. The extensor digitorum longus (EDL) muscle of six mice were exposed to lengthening contractions. Mice were anesthetized with 2% avertin (0.015 ml/kg) and supplemental doses were administered if the mouse responded to a toe pinch. Animals were placed on a plexiglass platform that was maintained at 37°C. The distal femur of the right hindlimb was fixed between screws and the foot was taped to the platform. The distal tendons of the EDL were exposed by incision and tied to the lever arm of a servomotor (Aurora Scientific, Richmond Hill ON, Canada) with 4-0 silk suture. Needle electrodes were placed adjacent to the peroneal nerve to stimulate dorsiflexor contraction (Grass Instruments, West Warwick, RI). Maximal isometric force (Po) was determined by stimulating the dorsiflexors maximally at optimum length (Lo) for force development. To induce muscle injury, 75 lengthening contractions were performed at 0.25 Hz for 5 min. For each lengthening contraction, the muscle was stimulated at 150 Hz and stretched 100 ms after the initiation of stimulation. Muscle stretch involved 20 % strain relative to Lo. Force deficit was evaluated 10 min after the 75 contractions and then the incision was closed with 7-0 silk suture. Animals were returned to their cage to recover until the time of sacrifice. Prior to sacrifice by cervical dislocation, animals were anesthetized with avertin and the EDL muscles were excised and flash frozen in liquid nitrogen. Muscles were stored in liquid nitrogen until RNA isolation. RNA Isolation. Total RNA was isolated from EDL muscles from control mice (n=3) and mice sacrificed at 6 h (n=3) and 72 h (n=3) after lengthening contractions according to the modified protocol of Chomzynski et al.. Frozen muscles were added to preweighed tubes containing TriReagent (Sigma-Aldrich, St. Louis, MO). Tissue was homogenized in TriReagent using a Tissue Tearor (Fischer, Pittsburgh, PA) at 30,000 rpm for 1 minute. Homogenates were transferred to sterile, RNAase free microcentrifuge tubes and incubated for 10 min at room temperature. Chloroform (0.2 ml; Sigma-Aldrich, St. Louis, MO) was added and after incubation, the samples were centrifuged for 15 min at 12,000 g and 4°C. Total RNA was precipitated from the aqueous phase by the addition of isopropanol (Sigma-Aldrich), and pelleted by centrifugation. Pellets were washed in ethanol (75 and 95 %) and air dried before resuspension in diethylpyrocarbonate (DEPC) treated water. RNA concentration was determined by optical density at 260 nm. III. Hybridization procedures and parameters: Microarray Hybridization and Analysis. To reduce measurement error, all membranes (Atlas mouse 1.2; Clontech, Palo Alto, CA) were prepared from the same lot. Additionally, each membrane was hybridized only once to avoid errors associated with stripping and multiple hybridizations. In order to minimize individual biological variability, total RNA was pooled from three animals for hybridization of arrays. Three arrays were used for the pooled sample at each time point. First strand cDNA probe generation from total RNA and microarray hybridization were performed according to manufacturer’s instructions (Atlas cDNA Expression Arrays User Manual, Clontech). 33P labeled cDNA was generated from sample RNA using [33P]-dATP (3000 Ci/mmol; ICN, Costa Mesa, CA) and Maloney murine leukemia virus (MMLV) reverse transcriptase (Clontech). Labeled probes were purified using nucleospin columns (Clontech). Array membranes were prehybridized in Express Hyb containing sheared Salmon testes DNA (Gibco BRL, Rockville, MD) for 30 min at 71°C in rotating hybridization tubes. Radiolabeled probe (3 x 106 cpm) was incubated in denaturing solution (Clontech) for 20 min at 71°C, after which Cot1 DNA/neutralizing solution was added and incubation continued for 10 min. The probe mixture was added to the hybridization tube and incubated with rotation overnight. Following hybridization, membranes were washed four times for 30 min in 2X saturated sodium citrate (SSC) and 1 % sodium dodecyl sulphate (SDS) at 71°C, once for 30 min in 0.1X SSC and 0.5% SDS at 71°C, and once for 5 min in 2X SSC at room temperature. Membranes were removed and immediately wrapped in plastic and exposed to a phosphor storage screen (Molecular Dynamics, Sunnyvale, CA) for four days. IV. Measurement data and specifications: Data Analysis. The membrane image was acquired using a Storm phosporimager (Molecular Dynamics) and Image Quant software. Array images were analyzed using Atlas Image 2.0 (Clontech) software to determine raw intensity levels of expression. Raw intensity data was imported into GeneSpring (Silicon Genetics, Redwood City, CA) expression analysis software. Intensity levels were normalized to the median expressed intensity on each of the arrays and averaged for the three arrays at each time point. Normalized expression levels for control arrays were established as a value of 1 and data from 6 and 72 h were expressed as fold changes relative to control. In order to simplify interpretation of the resulting dataset, a k-means cluster analysis was employed to group genes based on similarities in patterns of expression. To reduce the likelihood of false positives, the data was filtered using a criterion 2 fold change in expression prior to clustering. Miller et al. (61) have calculated that using an array of 10,000 features with a coefficient of variation (CV) of 20 percent, a 2 fold criterion for altered gene expression would result in zero false positives. The mean and median CV (15.8 and 14.3%, respectively) for all arrays in the current study fell within these guidelines for the elimination of false positives. Keywords: time-course
2004-01-27 | GSE992 | GEO
Project description:Among patients with intensive care unit-acquired weakness (ICUAW), skeletal muscle strength often decreases significantly. The present study aimed to explore the effects of testosterone propotionate on skeletal muscle using rat model of sepsis. Male SD rats were randomly divided into experimental group, model control group, sham operation group and blank control group. Rats in experimental group were given testosterone propionate two times a week, 10 mg/kg for 3 weeks. Maximal contraction force, fatigue index and cross-sectional area of the extensor digitorum longus (EDL) were measured. Myosin, IGF-1, p-AKT and p-mTOR levels in EDL were detected by Western blot. Histological changes of the testis and prostate were detected by hematoxylin and eosin staining. We found that maximal contraction force and fatigue index of EDL in experimental group were significantly higher than in model control group. Cross-sectional area of fast MHC muscle fiber of EDL in group was significantly higher than in model control group. The levels of myosin, IGF-1, p-AKT and p-mTOR of EDL in experimental group were significantly higher than in model control group. In addition, no testicle atrophy and prostate hyperplasia were detected in experimental group. In conclusion, these results suggest that testosterone propionate can significantly improve skeletal muscle strength, endurance and volume of septic rats, and the mechanism may be related to the activation of IGF-1/AKT pathway. Moreover, testosterone propionate with short duration does not cause testicular atrophy and prostate hyperplasia in septic rats. Therefore, testosterone propionate is a potential treatment for muscle malfunction in ICUAW patients.
| S-EPMC7000367 | biostudies-literature
Project description:The motor unit comprises a variable number of muscle fibres that connect through myelinated nerve fibres to a motoneuron (MN), the central drivers of activity. At the simplest level of organisation there exist phenotypically distinct MNs that activate corresponding muscle fibre types, but within an individual motor pool there typically exists a mixed population of fast and slow firing MNs, innervating groups of Type II and Type I fibres, respectively. Characterising the heterogeneity across multiple levels of motor unit organisation is critical to understanding changes that occur in response to physiological and pathological perturbations. Through a comprehensive assessment of muscle histology and ex vivo function, mathematical modelling and neuronal tracing, we demonstrate regional heterogeneities at the level of the MN, muscle fibre type composition and oxygen delivery kinetics of the rat extensor digitorum longus (EDL) muscle. Specifically, the EDL contains two phenotypically distinct regions: a relatively oxidative medial and a more glycolytic lateral compartment. Smaller muscle fibres in the medial compartment, in combination with a greater local capillary density, preserve tissue O2 partial pressure (PO2 ) during modelled activity. Conversely, capillary supply to the lateral compartment is calculated to be insufficient to defend active muscle PO2 but is likely optimised to facilitate metabolite removal. Simulation of in vivo muscle length change and phasic activation suggest that both compartments are able to generate similar net power. However, retrograde tracing demonstrates (counter to previous observations) that a negative relationship between soma size and C-bouton density exists. Finally, we confirm a lack of specificity of SK3 expression to slow MNs. Together, these data provide a reference for heterogeneities across the rat EDL motor unit and re-emphasise the importance of sampling technique.
| S-EPMC8930811 | biostudies-literature
Project description:I. Experimental Design: a. Type of experiment: Time course b. Experimental factors: 3 month old mice subjected to lengthening contractions of the extensor digitorum longus muscle (EDL). Samples of EDL collected at 6 and 72 h and compared to non-treated control. c. How many hybridizations in exp: 9 d. Common reference used for all hyb: no e. Quality control steps: All arrays used from same lot. Triplicate hybridizations performed for each time point. II. Samples used, extracts, preparation and labeling: Animals. Nine male C57BL/6 mice (3-4 mo of age, 27.8 ± 3.3 g body mass, Harlan Sprague-Dawley, Indianapolis, IN). Muscle Injury. The extensor digitorum longus (EDL) muscle of six mice were exposed to lengthening contractions. Mice were anesthetized with 2% avertin (0.015 ml/kg) and supplemental doses were administered if the mouse responded to a toe pinch. Animals were placed on a plexiglass platform that was maintained at 37°C. The distal femur of the right hindlimb was fixed between screws and the foot was taped to the platform. The distal tendons of the EDL were exposed by incision and tied to the lever arm of a servomotor (Aurora Scientific, Richmond Hill ON, Canada) with 4-0 silk suture. Needle electrodes were placed adjacent to the peroneal nerve to stimulate dorsiflexor contraction (Grass Instruments, West Warwick, RI). Maximal isometric force (Po) was determined by stimulating the dorsiflexors maximally at optimum length (Lo) for force development. To induce muscle injury, 75 lengthening contractions were performed at 0.25 Hz for 5 min. For each lengthening contraction, the muscle was stimulated at 150 Hz and stretched 100 ms after the initiation of stimulation. Muscle stretch involved 20 % strain relative to Lo. Force deficit was evaluated 10 min after the 75 contractions and then the incision was closed with 7-0 silk suture. Animals were returned to their cage to recover until the time of sacrifice. Prior to sacrifice by cervical dislocation, animals were anesthetized with avertin and the EDL muscles were excised and flash frozen in liquid nitrogen. Muscles were stored in liquid nitrogen until RNA isolation. RNA Isolation. Total RNA was isolated from EDL muscles from control mice (n=3) and mice sacrificed at 6 h (n=3) and 72 h (n=3) after lengthening contractions according to the modified protocol of Chomzynski et al.. Frozen muscles were added to preweighed tubes containing TriReagent (Sigma-Aldrich, St. Louis, MO). Tissue was homogenized in TriReagent using a Tissue Tearor (Fischer, Pittsburgh, PA) at 30,000 rpm for 1 minute. Homogenates were transferred to sterile, RNAase free microcentrifuge tubes and incubated for 10 min at room temperature. Chloroform (0.2 ml; Sigma-Aldrich, St. Louis, MO) was added and after incubation, the samples were centrifuged for 15 min at 12,000 g and 4°C. Total RNA was precipitated from the aqueous phase by the addition of isopropanol (Sigma-Aldrich), and pelleted by centrifugation. Pellets were washed in ethanol (75 and 95 %) and air dried before resuspension in diethylpyrocarbonate (DEPC) treated water. RNA concentration was determined by optical density at 260 nm. III. Hybridization procedures and parameters: Microarray Hybridization and Analysis. To reduce measurement error, all membranes (Atlas mouse 1.2; Clontech, Palo Alto, CA) were prepared from the same lot. Additionally, each membrane was hybridized only once to avoid errors associated with stripping and multiple hybridizations. In order to minimize individual biological variability, total RNA was pooled from three animals for hybridization of arrays. Three arrays were used for the pooled sample at each time point. First strand cDNA probe generation from total RNA and microarray hybridization were performed according to manufacturerâs instructions (Atlas cDNA Expression Arrays User Manual, Clontech). 33P labeled cDNA was generated from sample RNA using [33P]-dATP (3000 Ci/mmol; ICN, Costa Mesa, CA) and Maloney murine leukemia virus (MMLV) reverse transcriptase (Clontech). Labeled probes were purified using nucleospin columns (Clontech). Array membranes were prehybridized in Express Hyb containing sheared Salmon testes DNA (Gibco BRL, Rockville, MD) for 30 min at 71°C in rotating hybridization tubes. Radiolabeled probe (3 x 106 cpm) was incubated in denaturing solution (Clontech) for 20 min at 71°C, after which Cot1 DNA/neutralizing solution was added and incubation continued for 10 min. The probe mixture was added to the hybridization tube and incubated with rotation overnight. Following hybridization, membranes were washed four times for 30 min in 2X saturated sodium citrate (SSC) and 1 % sodium dodecyl sulphate (SDS) at 71°C, once for 30 min in 0.1X SSC and 0.5% SDS at 71°C, and once for 5 min in 2X SSC at room temperature. Membranes were removed and immediately wrapped in plastic and exposed to a phosphor storage screen (Molecular Dynamics, Sunnyvale, CA) for four days. IV. Measurement data and specifications: Data Analysis. The membrane image was acquired using a Storm phosporimager (Molecular Dynamics) and Image Quant software. Array images were analyzed using Atlas Image 2.0 (Clontech) software to determine raw intensity levels of expression. Raw intensity data was imported into GeneSpring (Silicon Genetics, Redwood City, CA) expression analysis software. Intensity levels were normalized to the median expressed intensity on each of the arrays and averaged for the three arrays at each time point. Normalized expression levels for control arrays were established as a value of 1 and data from 6 and 72 h were expressed as fold changes relative to control. In order to simplify interpretation of the resulting dataset, a k-means cluster analysis was employed to group genes based on similarities in patterns of expression. To reduce the likelihood of false positives, the data was filtered using a criterion 2 fold change in expression prior to clustering. Miller et al. (61) have calculated that using an array of 10,000 features with a coefficient of variation (CV) of 20 percent, a 2 fold criterion for altered gene expression would result in zero false positives. The mean and median CV (15.8 and 14.3%, respectively) for all arrays in the current study fell within these guidelines for the elimination of false positives.
2010-06-09 | E-GEOD-992 | biostudies-arrayexpress
Project description:INTRODUCTION:Because impaired excitation-contraction coupling and reduced sarcoplasmic reticulum (SR) Ca2+ release may contribute to the age-associated decline in skeletal muscle strength, we investigated the effect of aging on regulation of the skeletal muscle isoform of the ryanodine receptor (RyR1) by physiological channel ligands. METHODS:[3 H]Ryanodine binding to membranes from 8- and 26-month-old Fischer 344 extensor digitorum longus (EDL) and soleus muscles was used to investigate the effects of age on RyR1 modulation by Ca2+ and calmodulin (CaM). RESULTS:Aging reduced maximal Ca2+ -stimulated binding to EDL membranes. In 0.3 ?M Ca2+ , age reduced binding and CaM increased binding to EDL membranes. In 300 ?M Ca2+ , CaM reduced binding, but the age effect was not significant. Aging did not affect Ca2+ or CaM regulation of soleus RyR1. DISCUSSION:In aged fast-twitch muscle, impaired RyR1 Ca2+ regulation may contribute to lower SR Ca2+ release and reduced muscle function. Muscle Nerve 57: 1022-1025, 2018.
| S-EPMC5951731 | biostudies-literature
Project description:Recent studies of the ketogenic diet, an extremely high-fat diet with extremely low carbohydrates, suggest that it changes the energy metabolism properties of skeletal muscle. However, ketogenic diet effects on muscle metabolic characteristics are diverse and sometimes countervailing. Furthermore, ketogenic diet effects on skeletal muscle performance are unknown. After male Wistar rats (8 weeks of age) were assigned randomly to a control group (CON) and a ketogenic diet group (KD), they were fed for 4 weeks respectively with a control diet (10% fat, 10% protein, 80% carbohydrate) and a ketogenic diet (90% fat, 10% protein, 0% carbohydrate). After the 4-week feeding period, the extensor digitorum longus (EDL) muscle was evaluated ex vivo for twitch force, tetanic force, and fatigue. We also analyzed the myosin heavy chain composition, protein expression of metabolic enzymes and regulatory factors, and citrate synthase activity. No significant difference was found between CON and KD in twitch or tetanic forces or muscle fatigue. However, the KD citrate synthase activity and the protein expression of Sema3A, citrate synthase, succinate dehydrogenase, cytochrome c oxidase subunit 4, and 3-hydroxyacyl-CoA dehydrogenase were significantly higher than those of CON. Moreover, a myosin heavy chain shift occurred from type IIb to IIx in KD. These results demonstrated that the 4-week ketogenic diet improves skeletal muscle aerobic capacity without obstructing muscle contractile function in sedentary male rats and suggest involvement of Sema3A in the myosin heavy chain shift of EDL muscle.
| S-EPMC7598508 | biostudies-literature
Project description:AimSarcopenia - aging-related loss of muscle mass and muscle strength - is a key feature of the frailty model. In the present study, we aimed to elucidate the molecular biological changes associated with aging in the extensor digitorum longus muscle of senescence-accelerated mouse prone 8 mouse model by capillary electrophoresis-mass spectrometry.MethodsThree groups of senescence-accelerated mouse prone 8 mice were used, namely, 12-week-old (young; n = 5), 40-week-old (elderly; n = 5) and 55-week-old mice (late elderly; n = 5). The extensor digitorum longus muscle was collected. After preliminary analyses, metabolome analysis was carried out by capillary electrophoresis-mass spectrometry. Additionally, we examined whether the activity of enzymes in the metabolic pathway fluctuated with aging, by real-time polymerase chain reaction.ResultsAmong the 116 water-soluble metabolites associated with the central energy metabolism pathway, changes were observed in 19 metabolites between 12- and 40 -weeks-old, in 40 metabolites between 40- and 55-weeks-old, and in 57 metabolites between 12- and 55-weeks-old. The fluctuated metabolites that were common among the groups were Val, putrescine and His. The levels of putrescine, associated with cell proliferation, protein synthesis and nucleic acid synthesis, and β-Ala and His, a component of carnosine that is characterized by its anti-oxidant and anti-fatigue effects, decreased with age.ConclusionsWe confirmed that there were two aging-related metabolic changes in the extensor digitorum longus muscle of senescence-accelerated mouse prone 8 mice. Based on the changes in metabolites, cell senescence and fatigue in the extensor digitorum longus muscle might increase in old mice compared with those in young mice, showing molecular biological changes with aging. Geriatr Gerontol Int 2022; 22: 160-167.
| S-EPMC9302128 | biostudies-literature
Project description:Duchenne muscular dystrophy is due to genetic abnormalities in the dystrophin gene and represents one of the most frequent genetic childhood diseases. In the X-linked muscular dystrophy (mdx) mouse model of dystrophinopathy, different subtypes of skeletal muscles are affected to a varying degree albeit the same single base substitution within exon 23 of the dystrophin gene. Thus, to determine potential muscle subtype-specific differences in secondary alterations due to a deficiency in dystrophin, in this study, we carried out a comparative histological and proteomic survey of mdx muscles. We intentionally included the skeletal muscles that are often used for studying the pathomechanism of muscular dystrophy. Histological examinations revealed a significantly higher degree of central nucleation in the soleus and extensor digitorum longus muscles compared with the flexor digitorum brevis and interosseus muscles. Muscular hypertrophy of 20-25% was likewise only observed in the soleus and extensor digitorum longus muscles from mdx mice, but not in the flexor digitorum brevis and interosseus muscles. For proteomic analysis, muscle protein extracts were separated by fluorescence two-dimensional (2D) gel electrophoresis. Proteins with a significant change in their expression were identified by mass spectrometry. Proteomic profiling established an altered abundance of 24, 17, 19 and 5 protein species in the dystrophin-deficient soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscle, respectively. The key proteomic findings were verified by immunoblot analysis. The identified proteins are involved in the contraction-relaxation cycle, metabolite transport, muscle metabolism and the cellular stress response. Thus, histological and proteomic profiling of muscle subtypes from mdx mice indicated that distinct skeletal muscles are differentially affected by the loss of the membrane cytoskeletal protein, dystrophin. Varying degrees of perturbed protein expression patterns in the muscle subtypes from mdx mice may be due to dissimilar downstream events, including differences in muscle structure or compensatory mechanisms that counteract pathophysiological processes. The interosseus muscle from mdx mice possibly represents a naturally protected phenotype.
| S-EPMC3782555 | biostudies-literature