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PubMed1993

Single potassium channel currents activated by extracellular ATP in developing chick skeletal muscle: a role for second messengers.

1. In developing chick skeletal muscle, extracellular ATP activates an early excitatory current and a delayed potassium current. Previous work had shown that the potassium current elicited by ATP is sensitive to temperature and activates with a delay of nearly 1 s, suggesting that a second messenger is involved. The existence of a second messenger was confirmed by the observation that single potassium channels were activated in cell-attached patches, when ATP was applied outside of the patch pipette. 2. Two classes of ATP-activated potassium-channel currents were observed in cell-attached patches: one had a slope conductance of 23 pS, whereas the other had a slope conductance of 51 pS. 3. Pharmacological manipulations suggested that activation of the whole-cell potassium current by ATP did not require cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositol 1,4,5-triphosphate (IP3), nitric oxide, or a rise in internal free calcium. Additional pharmacological experiments suggested that activation of the whole-cell potassium current might not require activation of a G protein and probably did not involve intracellular protein phosphorylation. 4. The ability of arachidonic acid and its metabolites to activate potassium channels in chick skeletal muscle was also tested. Arachidonic acid, several prostaglandins and several leukotrienes activated whole-cell potassium currents. However, results with several inhibitors suggested that arachidonic acid and its metabolites are not necessary for activation of the whole-cell potassium current by ATP. 5. In excised outside-out membrane patches, ATP activated a single class of potassium channels. The slope conductance of these channels indicated that they are likely to be identical to the smaller of the two classes of second messenger activated potassium channels observed in cell-attached patches. 6. The observation that the larger class of potassium channels observed in cell-attached patches was absent in excised patches suggests that activation of these channels by ATP requires a cytosolic factor that is easily dialyzed away. In contrast, the observation that the smaller class of potassium channels could still be activated by ATP in excised patches suggests that the two classes of potassium channels are activated by different mechanisms. These results also indicate that all the molecules involved in coupling ATP receptor activation to opening of the smaller class of potassium channels remain closely associated with an excised patch. One possible explanation is that there might be an intramembranous second messenger.

Adenosine Triphosphate↗

PubMed1996

Synaptic integrins in developing, adult, and mutant muscle: selective association of alpha1, alpha7A, and alpha7B integrins with the neuromuscular junction.

Differentiation of both pre- and postsynaptic structures at the skeletal neuromuscular junction is organized by the basal lamina that occupies the synaptic cleft. As beta1 integrins are a major class of receptors for basal lamina components, we stained muscles with antibodies to the 10 integrin alpha subunits known to form dimers with beta1, to determine if any of these molecules were concentrated at synaptic sites on muscle fibers. In both developing and adult muscle, the integrin alpha1 chain was selectively associated with presynaptic cells (Schwann cells and/or nerve terminals), while alpha7 was present on both synaptic and extrasynaptic portions of the muscle fiber surface. Thus alpha1 and alpha7 integrins are present in synaptic membranes. Expression of the alpha7 chain was analyzed further by staining with antibodies specific for three alternatively spliced products of the alpha7 gene (A, B, and C), all of which were expressed in muscle. The alpha7A and alpha7B isoforms were confined to synaptic sites in adult muscle, while alpha7C was present both synaptically and extrasynaptically. In developing muscle, alpha7A appeared postnatally and specifically at the synapse; alpha7B was present throughout the muscle fiber perinatally, becoming confined to the synapse in the second postnatal week; and alpha7C was present extrasynaptically both perinatally and in adulthood. Thus, two of the alpha7 integrins are synapse-specific, and all three show distinct spatiotemporal patterns of expression within a single cell type. Finally, we asked whether perturbation of laminin expression affected the distribution of the alpha7 integrins. In normal mice, laminin beta2 is concentrated in synaptic basal lamina. In beta2-null mutant mice, alpha7A was still present at synaptic sites, but alpha7B was absent. This result provides genetic evidence that basal lamina composition is a determinant of integrin distribution.

Amino Acid Sequence↗

PubMed1984

Glycosphingolipids of chicken skeletal muscle in early development and genetic dystrophy.

The acidic and neutral GSL of chicken pectoral muscle and the activities of relevant sialyltransferase and glycosidases have been examined during embryonic and early post-hatching development. At this stage of myogenesis, a prominent shift to the neutral GSL of longer oligosaccharide length involving Forssman glycolipid most prominently and also globoside and GbOse3Cer occurred but the distribution of muscle-type gangliosides was not obviously affected. The glycosidase and sialyltransferase activities decreased dramatically just prior to or at hatching. The fusion-linked change in GSL suggests a role for terminal galactosamine and/or galactose residues in myoblast aggregation. A parallel developmental study of genetic muscular dystrophy revealed similar GSL levels and enzyme activities. A larger proportion of lactosylceramide in dystrophic muscle throughout development suggests a developmental lag in the mutant.

Aging↗

PubMed1989

Decrease in plasminogen activator correlates with synapse elimination during neonatal development of mouse skeletal muscle.

Previous studies have implicated proteases, acting extracellularly, in the mechanism of polyneuronal synapse elimination. Most studies have focused on mammalian, especially rodent, skeletal muscle, where retraction of subordinate nerve terminals occurs during a narrow time window 2-3 weeks after birth. To date no specific protease(s) has been detected that (i) coincides in time with maximal synapse elimination and (ii) is known to act extracellularly on specific extracellular matrix proteins. In previous studies of denervation in adult mouse muscle, rapid activation of urokinase-type plasminogen activator, a neutral serine protease, was detected. This enzyme, by activation of plasminogen to plasmin, specifically degrades matrix components such as fibronectin, type IV collagen, and laminin in muscle. We now present evidence for an initial increase and subsequent decrease in soluble urokinase-type PA--and, to a lesser extent, tissue PA--in developing muscle, suggesting postnatal developmental regulation of these enzymes during the period of maximal synapse elimination. Although considerably higher in specific activity, membrane-bound PA activity followed the wave of synapse elimination, possibly indicating a longer half-life of membrane-bound enzyme(s).

Aging↗

PubMed1974

The use of cytochalasin B to distinguish myoblasts from fibroblasts in cultures of developing chick striated muscle.

Cytochalasin B (5 mug/ml) elicits a differential effect on myoblasts and fibroblasts in culture. After 1 day in culture in the presence of the drug, two types of cells were observed, round cells and cells with elongated arms, designated "arborized" cells. Both cell types were examined in the electron microscope. The round cells contained aggregates of thin and thick filaments as well as a few intact sarcomeres. Within the arms of the arborized cells were bundles of intermediate sized filaments (100 A in diameter). The round cells could be shaken off the culture dish, washed free of cytochalasin B, and recultured to form myotubes. The remaining arborized cells lost their stellate shape when the drug was removed. The progeny of these cells gave rise to normal fibroblasts. Cytochalasin B, thus could be used to identify and isolate myoblasts prior to their fusion into developing muscle. It is suggested that this differential effect of the drug can be used to prepare pure cultures of fusible muscle cells uncontaminated by fibroblasts.

Animals↗

PubMed1994

The role of anthropometric characteristics in modern pentathlon performance in female athletes.

In order to determine the role of body build characteristics in modern pentathletes, 65 female participants at the IXth World Modern Pentathlon Championships, 1989 (Wiener Neustadt, Austria) were investigated. Of these, 54 participate in the competition; the other 11 were reserves. Their mean (+/- S.D.) age was 22.34 +/- 3.97 years (range 16.08-32.58 years). Anthropometric characteristics (body mass, lengths, breadths, girths and skinfolds), somatotype and body composition estimates were determined. Compared with other female athletes (e.g. swimmers, runners, fencers), the elite modern pentathletes were rather tall (x = 168.1 cm) and, as indicated by the body mass index (x = 21.6), they had a high mass relative to their stature. Based on skinfolds, body fat was estimated as 16%, and anthropometrically determined somatotype was on average 2.5-3.9-2.8. The relationship between the athletes' anthropometric characteristics and modern pentathlon performance was investigated by means of Pearson zero-order correlations between the physical traits and the competitive performance scores. Significant correlations were found mainly for the 'fat' variables, such as skinfolds, percent fat and the endomorphy component, r varying from -0.34 to -0.58, which indicated an inverse relationship between the amount of fatness and modern pentathlon performance. To investigate this relationship further, Pearson zero-order correlations were calculated between the respective factors scores, derived from a rotated factor pattern (Varimax) carried out on selected somatic variables, and the performance scores. The results demonstrated that, in relation to anthropometric characteristics, modern pentathlon performance in females is mainly associated with the 'fat development' factor, and to a lesser degree with the 'bone-muscle development' factor. Variables representing linearity of physique seem to be unrelated to modern pentathlon performance. Stepwise regression analysis revealed that 42.4% of the variance in modern pentathlon performance can be explained by the following anthropometric variables: sum of 10 skinfolds, biacromial breadth and humerus diameter. To obtain a high level of modern pentathlon performance, it would seem that a female athlete must above all have a low level of body fatness and, to a lesser degree, a relatively high level of lean body mass.

Adolescent↗

PubMed1987

[Succinate dehydrogenase activity of brachial muscle antagonists after decrease in the force load at various times of the day in rats].

In 140 male rats of Wistar strain the distal part of one thoracic extremity has been amputated in such a way that places of the brachial muscle fixation remained intact. This resulted in decrease of the force component of muscle contractions (more intensive in the musculus triceps brachii) without essential swing and speed of movements. The operation was performed during one day at three series: at 7 a. m. 3 p. m. and 11 p. m. During following 45 days the muscles developing various contraction force were studied: the medial head of the musculus triceps brachii and brachial muscle. Succinate dehydrogenase (SDG) activity in muscle fibers (MF) was estimated photometrically. In MF initially characterized with various SDG activity, attention was payed to its dynamics and also relation of the fibers number, possessing various level of the enzymatic activity was taken into consideration. SDG activity in MF and relation of various MF change wavy-like, fluctuating from the control meaning and returning to them again. MF with different SDG activity do not similarly react to a sharp decrease of the force loading. Fibers with high enzymatic activity react to changes of the force loading immediately, intensity of the reaction at this stage slightly depends on degree of biomechanical changes; at later stages the response is differentiated: if the loading is decreased considerably (musculus triceps brachii), the process continues developing and increasing uninterruptedly, if it decreases moderately (brachial muscle)--it stabilizes and even has tendency to adaptation and returning towards the control state.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1975

Relationship of the sarcoplasmic reticulum to fibril and triadic junction development in skeletal muscle fibers of fetal monkeys and humans.

Examinations of stages of fibril development in muscle fibers of seven Rhesus monkey and six human fetuses reveal SR tubules encircling the Z lines at all stages of fibril development. The encircling SR tubules are continuous with the SR network of tubules which is found surrounding fibrils at all stages of development observed. The SR tubules encircling the Z lines show connections (electron-opaque strands) with the Z lines. The developing triadic junction shows a progressive increase in complexity of structures within the junction. First, membranes of T and SR become apposed with no visible structure between them- Second, tenuous connections are found traversing the space between apposed membranes. Third, well developed bridges are seen traversing the space. And finally, an intermediate density midway between the apposed membranes and parallel to them is found in favorable sections. Junctions between T tubule membranes were also observed and the structures in these junctions are somewhat similar to those found in junctions between T and SR membranes. The change in orientation of triads from predominantly longitudinal to predominantly transverse is complete in the 18-week monkey fetus and incomplete in the latest stage (28-week) of fetal development observed in humans.

Animals↗

PubMed1997

Tubulin tyrosine ligase: protein and mRNA expression in developing rat skeletal muscle.

Alpha tubulin can be post-translationally tyrosinated at the carboxy-terminus by a specific enzyme: tubulin tyrosine ligase. The expression of tubulin tyrosine ligase mRNA and protein during the development of rat skeletal muscle was examined in the present study. A portion of the coding region of the rat ligase cDNA was isolated and sequenced. The nucleotide and amino acid sequences showed about 90% homology with previously reported porcine and bovine ligase sequences. In newborn rats, ligase mRNA and protein were highly expressed in skeletal muscle. During early postnatal development, however, both ligase mRNA and protein dropped down dramatically. Quantitative measurements revealed that ligase protein at postnatal day 20 represented only 10% or less of the level at postnatal day 1. Ligase mRNA expression was also examined during the myogenesis in vitro. A strong ligase mRNA signal was detected in both undifferentiated myoblasts and cross-striated, contractile myotubes. The present results suggest that, during muscle differentiation, ligase function may be regulated by the amount of available mRNA. The discrepancy in the ligase expression between the in vivo and in vitro myogenesis suggests that factors controlling the levels of mRNA in vivo are lost in vitro.

Amino Acid Sequence↗

PubMed2003

Mitochondrial distribution and glycogen dynamics suggest diffusion constraints in muscle fibers of the blue crab, Callinectes sapidus.

The scaling of mitochondrial distribution, citrate synthase activity, and post-contractile glycogen recovery was examined in muscle fibers of the blue crab, Callinectes sapidus. The fast-twitch muscle fibers of C. sapidus can reach extremely large dimensions, which may impose constraints on aerobic metabolic processes. However, muscle cells from small crabs are not giant, meaning that during development muscle fibers cross and greatly exceed the surface area to volume (SAV) and diffusion threshold that is adhered to by the cells of most organisms. Cell diameters in the smallest size class were approximately 100 microm, while the largest size class had cell diameters in excess of 500 microm. In the smallest cells, the fractional area of subsarcolemmal and intermyofibrillar mitochondria was similar. However, in the largest cells, mitochondria were almost exclusively subsarcolemmal. Total fractional area of mitochondria was highest in the largest cells due to a proliferation of subsarcolemmal mitochondria. In contrast, citrate synthase activity decreased as cell size increased. Following burst contractile activity, glycogen concentrations decreased significantly and remained depressed for several hours in muscle comprised of giant cells, consistent with previous findings that anaerobic glycogenolysis fuels certain components of post-contractile recovery. However, in muscle composed of the smallest muscle cells, glycogen levels did not decrease significantly following burst activity. While normal scaling of aerobic metabolism would predict a slower aerobic recovery in larger animals, the present results suggest that cellular organization, SAV, and intracellular diffusion distances also impose constraints on aerobic processes in C. sapidus.

Animals↗

PubMed1987

Transient appearance of a fast myosin heavy chain epitope in slow-type muscle fibres during stretch hypertrophy of the anterior latissimus dorsi muscle in the adult chicken.

Myosin expression during hypertrophy of the chicken anterior latissimus dorsi (ALD) muscle was investigated by immunocytochemical procedures using monoclonal antibodies to the fast and slow isoforms of the myosin heavy chain (myosin HC). Antifast antibody 1F9 bound to the adult fast HC of pectoralis muscle and cross-reacted with the HC found in early developing muscle. Antislow antibody 3D1 bound exclusively to the HC of slow myosin 2 (SM2). Stretch hypertrophy of the ALD was produced by attaching a weight to the wing; there was no evidence for a change in fibre number in the muscle. Between 4 and 6 days of stretch there appeared a dramatic increase in the number of fibres staining with the antifast antibody which reached a peak between 12 and 19 days. By this time between 28 and 52% of the fibres in the stretched ALD stained to varying degrees with the antifast antibody compared with much less than 1% in the unstretched control ALD. Most antifast-stained fibres in the stretched muscle also stained with the antislow antibody; the contralateral control muscle showed mostly antislow staining except for the very small number of strongly antifast-stained fibres. By 50 days in some birds and by 80 days in all birds antifast staining had returned to normal. Analysis of the isomyosin composition of the ALD by native gel electrophoresis did not reveal a significant increase in fast myosin content of the hypertrophied muscle even though immunocytochemical staining may have suggested otherwise.

Animals↗

PubMed2002

Adverse and beneficial functions of proteolytic enzymes in skeletal muscle. An overview.

Proteolytic enzymes (proteases) comprise a family of enzymes which hydrolyse protein or peptide substrates in the generalised process of intracellular protein degradation, a process essential for the normal functioning of all cells. Proteases may also have a wide range of additional functions, including metabolic control of physiologically active oligopeptides or precursor protein forms, antigen presentation/recognition by the major histocompatibility complex in the cellular immune response, as well as in digestion, blood clotting, complement activation, etc. In this article, the nomenclature and classification of proteolytic enzymes in skeletal muscle, and their role in normal muscle physiological processes have been reviewed, including exercise, muscle development and ageing. Although proteases play an important role in normal muscle functioning, in pathological situations the enzymes may themselves be regarded as 'toxic agents' in terms of their damaging effects on muscle tissue. Muscle damage resulting from inappropriate activity of proteolytic enzymes in muscle wasting associated with muscular dystrophies, denervation atrophy, inflammatory myopathies, cancer, sepsis, diabetes and alcoholism have been reviewed. In addition, evidence that the adverse effects of drugs known to induce muscle wasting, such as corticosteroids, (or beneficial effects of growth promoting drugs) may be mediated via proteolytic enzymes is also reviewed.

Adrenal Cortex Hormones↗

PubMed2006

The denervated muscle: facts and hypotheses. A historical review.

Denervation changes in skeletal muscle (atrophy; alterations of myofibrillar expression, muscle membrane electrical properties, ACh sensitivity and excitation-contraction coupling process; fibrillation), and their possible causes are reviewed. All changes can be counteracted by muscle electrostimulation, while denervation-like effects can be caused by the complete conduction block in muscle nerve. These results do not support the hypothesis that the lack of neurotrophic, non-motor factors plays a role in denervation phenomena. Instead they support the view that the lack of neuromotor discharge is the only cause of the phenomena and that neuromotor activity is an essential factor in regulating muscle properties. However, some experimental results cannot apparently be explained by the lack of neuromotor impulses, and may still suggest that neurotrophic influences exist. A hypothesis is that neurotrophic factors, too feeble to maintain a role in completely differentiated, adult muscles, can concur with neuromotor activity in the differentiation of immature, developing muscles.

Animals↗

PubMed1980

Canine gastric sling fibers: contractile properties.

The contractile properties of canine sling fibers and the longitudinal and circular muscles were characterized and compared. The longitudinal and circular muscles developed spontaneous contractions, but the sling fibers developed no spontaneous phasic contractions. The cholinergic receptors of all three muscles were excitatory. The alpha- and beta-adrenergic receptors of the longitudinal and circular muscles were inhibitory, but the sling fiber alpha-adrenergic receptors were excitatory and the beta receptors inhibitory. Large concentrations of atrophine induced contractions of all three muscles. Some of the contractile properties of the sling fibers are more similar to those of the distal esophageal circular muscle than to those of the gastric longitudinal and circular muscles. The sling fibers together with the esophageal circular muscle may play an important role in the gastroesophageal sphincter mechanism.

Acetylcholine↗

PubMed2006

Nuclear targeting of the CaMKII anchoring protein alphaKAP is regulated by alternative splicing and protein kinases.

alphaKAP is an anchoring protein for the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and is encoded within the same gene as the CaMKIIalpha isoform. alphaKAP co-assembles with CaMKII and targets such heteromers to the membrane of the sarcoplasmic reticulum, where CaMKII can regulate Ca(2+) homeostasis. CaMKII has also nuclear functions in skeletal muscle, however, the nuclear targeting mechanism has been elusive. We show here that developmentally regulated splicing of exon Ealpha(B) generates a functional nuclear localization signal (NLS) in alphaKAP(B), the dominant alphaKAP variant in mature muscle. The alphaKAP(A) variant lacks the NLS and dominates in developing muscle before and around birth. Both alphaKAP variants localize to membranes, but a small fraction of alphaKAP(B) is additionally found in the nucleus. Indeed, alpha-karyopherins that mediate nuclear import bound to alphaKAP(B) but not alphaKAP(A) in vitro. When the N-terminal membrane anchor of alphaKAP was deleted, localization of alphaKAP(B) but not alphaKAP(A) became predominantly nuclear. Co-expression of constitutively active CaMKI and IV, which do not bind to alphaKAP, interfered with nuclear localization of alphaKAP(B). CaMKIIalpha was found essentially exclusively in the cytoplasm when expressed in cell lines but was targeted to the nucleus when co-expressed with the nuclear form of alphaKAP(B). Thus, nuclear targeting of cytoplasmic CaMKII isoforms by alphaKAP may be regulated by developmentally controlled alternative splicing and by protein kinases.

Alternative Splicing↗

PubMed2004

Large-scale gene expression analysis of human skeletal myoblast differentiation.

To study pathways involved in human skeletal myogenesis, we profiled gene expression in human primary myoblast cells derived from three individuals using both oligonucleotide and cDNA microarrays. Following stringent statistical testing (false-positive rate 0.4%), we identified 146 genes differentially expressed over time. Interestingly, 86 of these genes have not been reported to be involved in myogenesis in mouse cell lines. This demonstrates the additional value of human primary cell cultures in the study of muscle differentiation. Many of the identified genes play a role in muscle regeneration, indicating the close relationship of this process with muscle development. In addition, we found overlap with expression profiling studies in muscle from Duchenne muscular dystrophy patients, confirming ongoing muscle regeneration in Duchenne muscular dystrophy. Further study of these genes can bring new insights into the process of muscle differentiation, and they are candidate genes for neuromuscular disorders with an as yet unidentified cause.

Cell Differentiation↗

PubMed2003

Continuous, intermitted and sporadic motor unit activity in the trapezius muscle during prolonged computer work.

The Cinderella hypothesis postulates the continuous activity of specific motor units (MUs) during low-level muscle contraction. The MUs may become metabolically overloaded, with the subject developing muscle pain and strain. The hypothesis requires MUs that are active for a time long enough to actually damage muscle fibers. The aim of this study was to determine if there are continuously active MUs in the right trapezius muscle during normal computer work using a computer mouse. Fourteen healthy subjects executed an interactive computer-learning program (ErgoLight) for 30 min. Six-channel intramuscular EMG and two-channel surface EMG signals were recorded from two positions of the trapezius muscle. Decomposition was achieved with automated, multi-channel, long-term decomposition software (EMG-LODEC). In two out of the 14 subjects, three MUs were continuously active throughout the 30 min. Although the majority of the MUs were active during only part of the experimental session, an ordered on-off behavior (e.g. substitution) pattern was not observed. As long-lasting activity was verified in some subjects, the results support the Cinderella hypothesis. However, it cannot be concluded here how long the MUs could stay active. If continuous activity overloads low threshold MUs, the potential exists for selective fibre injuries in low threshold MUs of the trapezius muscle in subjects exposed to long-term computer work.

Adult↗

PubMed2003

Differences in the function of three conserved E-boxes of the muscle creatine kinase gene in cultured myocytes and in transgenic mouse skeletal and cardiac muscle.

The 1256-base pair enhancer-promoter of the mouse muscle creatine kinase gene includes three CAnnTG E-boxes that are conserved among mammals and have flanking and middle sequences conforming to consensus muscle regulatory factor binding sites. This study seeks to determine whether these E-boxes are critical for muscle creatine kinase expression in physiologically distinct muscles. Mutations of the "right" and "left" E-boxes in the enhancer decreased expression in cultured skeletal myocytes approximately 10- and 2-fold, respectively, whereas a "promoter" E-box mutation had little effect. In neonatal myocardiocytes, the left E-box mutation decreased expression approximately 3-fold, whereas right or promoter E-box mutations had no effect. Very different effects were seen in transgenic mice, where the promoter E-box mutation decreased expression in quadriceps, extensor digitorum longus, and soleus approximately 10-fold, and approximately 100-fold in distal tongue, diaphragm, and ventricle. The right E-box mutation, tested in the presence of the other two mutations, caused a significant decrease in distal tongue, but not in quadriceps, extensor digitorum longus, soleus, or ventricle. Mutation of the left E-box actually raised expression in soleus, suggesting a possible repressor role for this control element. The discrepancies between mutation effects in differentiating skeletal muscle cultures, neonatal myocardiocytes, and adult mice suggested that the E-boxes might play different roles during muscle development and adult steady-state function. However, transgenic analysis of embryonic and early postnatal mice indicated no positive role for these three E-boxes in early development, implying that differences in E-box function between adult muscle and cultured cells are the result of physiological signals.

Animals↗