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In many tissues, actin monomers polymerize into actin (thin) filaments of precise lengths. Although the exact mechanisms involved remain unresolved, it is proposed that 'molecular rulers' dictate the lengths of the actin filaments. The giant nebulin molecule is a prime candidate for specifying thin filament lengths in striated muscle, but this idea has never been proven. To test this hypothesis, we used RNA interference technology in rat cardiac myocytes. Live cell imaging and triple staining revealed a dramatic elongation of the preexisting thin filaments from their pointed ends upon nebulin knockdown, demonstrating its role in length maintenance; the barbed ends were unaffected. When the thin filaments were depolymerized with latrunculin B, myocytes with decreased nebulin levels reassembled them to unrestricted lengths, demonstrating its importance in length specification. Finally, knockdown of nebulin in skeletal myotubes revealed its involvement in myofibrillogenesis. These data are consistent with nebulin functioning as a thin filament ruler and provide insight into mechanisms dictating macromolecular assembly.

The larval and polyp stages of extant Cnidaria are bi-layered with an absence of mesoderm and its differentiation products. This anatomy originally prompted the diploblast classification of the cnidarian phylum. The medusa stage, or jellyfish, however, has a more complex anatomy characterized by a swimming bell with a well-developed striated muscle layer. Based on developmental histology of the hydrozoan medusa this muscle derives from the entocodon, a mesoderm-like third cell layer established at the onset of medusa formation. According to recent molecular studies cnidarian homologs to bilaterian mesoderm and myogenic regulators are expressed in the larval and polyp stages as well as in the entocodon and derived striated muscle. Moreover striated and smooth muscle cells may have evolved directly and independently from non-muscle cells as indicated by phylogenetic analysis of myosin heavy chain genes (MHC class II). To accommodate all evidences we propose that striated muscle-based locomotion coevolved with the nervous and digestive systems in a basic metazoan Bauplan from which the ancestors of the Ctenophora (comb jellyfish), Cnidaria (jellyfish and polyps), as well as the Bilateria are derived. We argue for a motile tri-layered cnidarian ancestor and a monophyletic descent of striated muscle in Cnidaria and Bilateria. As a consequence, diploblasty evolved secondarily in cnidarian larvae and polyps. Keywords: Basic Bauplan; bHLH gene; Cnidaria; Entocodon; Hydrozoan jellyfish; Medusa; Mesoderm; Metazoan evolution; Monophyletic descent; Origin of triploblasty; Prebilaterian ancestor; Striated muscle; Zootype

Skeletal muscles are diverse in their properties, with specific contractile characteristics being matched to particular functions. In this study, published values of contractile properties for >130 diverse skeletal muscles were analyzed to detect common elements that account for variability in shortening velocity and force production. Body mass was found to be a significant predictor of shortening velocity in terrestrial and flying animals, with smaller animals possessing faster muscles. Although previous studies of terrestrial mammals revealed similar trends, the current study indicates that this pattern is more universal than previously appreciated. In contrast, shortening velocity in muscles used for swimming and nonlocomotory functions is not significantly affected by body size. Although force production is more uniform than shortening velocity, a significant correlation with shortening velocity was detected in muscles used for locomotion, with faster muscles tending to produce more force. Overall, the contractile properties of skeletal muscles are conserved among phylogenic groups, but have been significantly influenced by other factors such as body size and mode of locomotion. skeletal muscle; scaling; shortening velocity; tetanic tension

ATP is well known for its role as an intracellular energy source. However, there is increasing awareness of its role as an extracellular messenger molecule (Burnstock, 1997). Although evidence for the presence of receptors for extracellular ATP on skeletal myoblasts was first published in 1983 (Kolb and Wakelam), their physiological function has remained unclear. In this paper we used primary cultures of rat skeletal muscle satellite cells to investigate the role of purinergic signaling in muscle formation. Using immunocytochemistry, RT-PCR, and electro-physiology, we demonstrate that the ionotropic P2[X.sub.5] receptor is present on satellite cells and that activation of a P2X receptor inhibits proliferation, stimulates expression of markers of muscle cell differentiation, including myogenin, p21, and myosin heavy chain, and increases the rate of myotube formation. Furthermore, we demonstrate that ATP application results in a significant and rapid increase in the phosphorylation of MAPKs, particularly p38, and that inhibition of p38 activity can prevent the effect of ATP on cell number. These results not only demonstrate the existence of a novel regulator of skeletal muscle differentiation, namely ATP, but also a new role for ionotropic P2X receptors in the control of cell fate.

Leptin increases FA oxidation in lean but not obese human skeletal muscle: evidence of peripheral leptin resistance. Am J Physiol Endocrinol Metab 283: E187-E192, 2002. First published March 19, 2002; 10.1152/ajpendo.00542.2001.--The adipocyte-derived hormone leptin has been shown to acutely increase fatty acid (FA) oxidation and decrease esterification in resting rodent skeletal muscle. However, the effects of leptin on human skeletal muscle FA metabolism are completely unknown. In these studies, we have utilized an isolated human skeletal muscle preparation combined with the pulse-chase technique to measure FA metabolism with and without leptin in lean and obese human skeletal muscle. Under basal conditions (in the absence of leptin), muscle from the obese demonstrated significantly elevated levels of total FA uptake (+72%, P = 0.038) and enhanced rates of FA esterification into triacylglycerol (+102%, P = 0.042) compared with lean subjects. In the presence of leptin, lean muscle had elevated rates of endogenous (+103%, P = 0.01) and exogenous (+150%, P = 0.03) palmitate oxidation. When the ratio of esterification to exogenous oxidation was examined, leptin reduced this ratio (-47%, P = 0.032), demonstrating the increased partitioning of FA toward oxidation and away from storage. Contrary to these findings in lean muscle, leptin had no effect on FA metabolism in skeletal muscle of the obese. This study provides the first evidence that leptin increases FA oxidation in skeletal muscle of lean, but not obese humans, thus demonstrating the development of leptin resistance in obese human skeletal muscle. pulse-chase technique; triacylglycerol; rectus abdominus

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microcoscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD[34.sup.+] and CD[45.sup.-] fraction. Cells in this fraction were ~94% positive for Sca-1, and mostly negative (

Carnitine palmitoyltransferase I (CPT I), which is expressed as two distinct isoforms in liver ([alpha]) and muscle ([beta]), catalyzes the rate-limiting step in the transport of fatty acid into the mitochondria. Malonyl-CoA, a potent inhibitor of CPT I, is considered a key regulator of fatty acid oxidation in both tissues. Still unanswered is how muscle [beta]-oxidation proceeds despite malonyl-CoA concentrations that exceed the I[C.sub.50] for CPT I[beta]. We evaluated malonyl-CoA-suppressible [[sup.14]C]palmitate oxidation and CPT I activity in homogenates of red (RG) and white (WG) gastrocnemius, soleus (SOL), and extensor digitorum longus (EDL) muscles. Adding 10 [micro]M malonyl-CoA inhibited palmitate oxidation by 29, 39, 60, and 89% in RG, SOL, EDL, and WG, respectively. Thus malonyl-CoA resistance, which correlated strongly (0.678) with absolute oxidation rates (RG > SOL > EDL > WG), was greater in red than in white muscles. Similarly, malonyl-CoA-resistant palmitate oxidation and CPT I activity were greater in mitochondria from RG compared with WG. Ribonuclease protection assays were performed to evaluate whether our data might be explained by differential expression of CPT I splice variants. We detected the presence of two CPT I[beta] splice variants that were more abundant in red compared with white muscle, but the relative expression of the two mRNA species was unrelated to malonyl-CoA resistance. These results provide evidence of a malonyl-CoA-insensitive CPT I activity in red muscle, suggesting fiber type-specific expression of distinct CPT I isoforms and/or posttranslational modulations that have yet to be elucidated. fatty acid oxidation; fiber-type specificity

The purpose of this study was to determine if age-related muscle atrophy is associated with an increased rate of protein degradation in extensor digitorum longus (EDL) muscles from young (YG; 2-4 months), middle-aged (MA; 12-17 months), and aged (AG; 22-24 months) B6C3F1 mice. EDL muscles from AG mice weighed less than EDL muscles from MA mice (p = .01). EDL muscles from MA mice weighed more than EDL muscles from YG mice (p = .02). The rate of protein degradation, as assessed by tyrosine release during in vitro incubations, was higher in EDL muscles from AG mice than it was in those from MA mice (p = .03). The rate of protein degradation was higher in EDL muscles from YG mice than it was in those from MA mice (p = .04). An inverse relationship existed between muscle mass and protein degradation (r = -.67; p = .0001). We conclude that skeletal muscle protein degradation rates decrease with maturation and increase with advancing age.

Considerable evidence argues that consumption of beef products from cattle infected with bovine spongiform encephalopathy (BSE) prions causes new variant Creutzfeldt-Jakob disease. In an effort to prevent new variant Creutzfeldt-Jakob disease, certain 'specified offals,' including neural and lymphatic tissues, thought to contain high titers of prions have been excluded from foods destined for human consumption [Phillips, N. A., Bridgeman, J. & Ferguson-Smith, M. (2000) in The BSE Inquiry (Stationery Office, London), Vol. 6, pp. 413-451]. Here we report that mouse skeletal muscle can propagate prions and accumulate substantial titers of these pathogens. We found both high prion titers and the disease-causing isoform of the prion protein (Pr[P.sup.Sc]) in the skeletal muscle of wild-type mice inoculated with either the Me7 or Rocky Mountain Laboratory strain of murine prions. Particular muscles accumulated distinct levels of Pr[P.sup.Sc], with the highest levels observed in muscle from the hind limb. To determine whether prions are produced or merely accumulate intramuscularly, we established transgenic mice expressing either mouse or Syrian hamster PrP exclusively in muscle. Inoculating these mice intramuscularly with prions resulted in the formation of high titers of nascent prions in muscle. In contrast, inoculating mice in which PrP expression was targeted to hepatocytes resulted in low prion titers. Our data demonstrate that factors in addition to the amount of PrP expressed determine the tropism of prions for certain tissues. That some muscles are intrinsically capable of accumulating substantial titers of prions is of particular concern. Because significant dietary exposure to prions might occur through the consumption of meat, even if it is largely free of neural and lymphatic tissue, a comprehensive effort to map the distribution of prions in the muscle of infected livestock is needed. Furthermore, muscle may provide a readily biopsied tissue from which to diagnose prion disease in asymptomatic animals and even humans.

Effect of a high-salt diet on oxidant enzyme activity in skeletal muscle microcirculation. Am J Physiol Heart Circ Physiol 282: H395-H402, 2002. First published October 11, 2001; 10.1152/ajpheart.00354.2001.--Increased salt intake attenuates the endothelium-dependent dilation of skeletal muscle arterioles by abolishing local nitric oxide (NO) activity. There is evidence of oxidative stress in arteriolar and venular walls of rats fed a high-salt diet, and depressed arteriolar responses to acetylcholine (ACh) in these rats are reversed by scavengers of reactive oxygen species (ROS). In this study, we tested the hypothesis that this salt-dependent increase in microvascular ROS and the resulting attenuation of endothelium-dependent dilation are due to increased expression and/or activity of oxidant enzymes in the microvascular wall. Resting arteriolar and venular wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction, was consistently higher in the spinotrapezius muscle of rats fed a high-salt diet (7% NaCl, HS) for 4-5 wk than in those fed a normal diet (0.45% NaCl, NS) for the same duration. Western analysis of protein from isolated microvessels showed no difference between HS and NS rats in the expression of NAD(P)H oxidase or xanthine oxidase. Inhibition of NAD(P)H oxidase and/or xanthine oxidase with diphenyleneiodonium chloride and oxypurinol, respectively, reduced resting arteriolar wall oxidant activity to normal levels in HS rats but had no effect in NS rats, suggesting that the basal activities of NAD(P)H oxidase and xanthine oxidase are increased in HS microvessels. However, inhibition of these enzymes in HS rats did not restore normal arteriolar responses to ACh, suggesting that this stimulus activates an alternate source of ROS that eliminates the role of NO in the subsequent dilation. dietary salt; reactive oxygen species; nitric oxide; endothelium-dependent dilation

The ratio of skeletal muscle to adipose tissue-free body mass (SM/ATFM) was examined in a large cohort of healthy adults, including 139 men and 165 women. The effect of disease on the SM/ATFM ratio was also explored with a group of AIDS patients.

Functional inactivation of the IGF-I and insulin receptors (IGF-IR and IR) in striated muscle has been found to cause type 2 diabetes. A transgenic mouse with a dominant-negative insulin-like growth factor-1 receptor specifically targeted to the striated muscle was developed for study. The mice, MKR mice, were excellent as a model for this type of study. They showed striking loss of function of the IR and the IGF-IR

The authors present their research into the steroid receptor coactivator, GRIP-1, and its necessary function in MEF-2C-dependent gene expression and skeletal muscle differentiation.

In many bilaterian animals members of the Otx gene family are expressed in head or brain structures. Cnidarians, however, have no clearly homologous head and no distinct brain; but an Otx homolog from the jellyfish Podocoryne carnea is highly conserved in sequence and domain structure. Sequence similarities extend well beyond the homeodomain and Podocoryne Otx can be aligned over its entire length to human OTX1, OTX2, and CRX. The overall structure of Otx is better conserved from Podocoryne to deuterostomes while protostomes appear to be more derived. In contrast, functions seem to be conserved from protostomes to vertebrates but not in Podocoryne or echinoderms. Podocoryne Otx is expressed only during medusa bud formation and becomes restricted to the striated muscle of medusae. Cnidaria are the most basal animals with striated muscle. Podocoryne polyps have no striated muscle and no Otx expression; both appear only during the asexual medusa budding process. The common ancestor of all animals that gave rise to cnidarians, protostomes, and deuterostomes already had an Otx gene more similar to today's Podocoryne and human homologs than to Drosophila otd, while the head-specific function appears to have evolved only later.

Connectin, or titin, is a giant muscle protein which is important for spatial and temporal control of sarcomeres found in striated muscle. Titin kinase is the only catalytic domain of titin, and its crystal structure shows the way in which a tyrosine inhibits the active site. This tyrosine appears to be a binding partner of the substrate of titin kinase. The research shows that the titin kinase may be important in myofibrillogenesis.

Research was conducted to examine the effects of the hemoglobin-based oxygen carrier diaspirin-cross-linked hemoglobin (DCLHb) in an ischemia-reperfusion model of hamster striated skin muscle. Microcirculation and local tissue analysis of the dorsal skinfold chamber was performed by a multiwire platinum surface electrode and intravital fluorescence microscopy. Results suggest the possibility that alterations of leukocyte-endothelial cell interactions may explain the conclusion that DCLHb attenuates postischemic reperfusion injury of striated skin muscle.

Research indicates that the biochemical vascular endothelial growth factor (VEGF) is directly involved in the regulation of vascular density in mammalian skeletal muscles. Immunohistochemical experiments using a rat chronic motor nerve glycolytic skeletal muscle model are described. Innate glycolytic muscles contain less VEGF than oxidative muscles.

The structural regions of the cardiac channel alpha sub(1C) subunit involved in calcium-dependent inactivation was probed through analyzing a series of chimeric alpha1 subunits wherein the alpha sub(1S) replaced the major cytoplasmic domains of alphasub(1) channels. Results have indicated that the cytoplasmic carboxyl terminus and the I-II/II-III loops mediated the molecular mechanism of calcium-dependent inactivation.

Research was conducted to examine the relationships between activation and contraction and between length and tension of the cricopharyngeus (CP), the primary muscle of the upper esophageal sphincter. Using 40 decerebrate cats, the resting and active tension of the CP and CP force and electromyographic (EMG) activity were recorded simultaneously during electrical stimulation of the pharyngeoesophageal (PE) nerve, esophageal distension or swallowing. Results show that the CP exhibits tension throughout its physiological range of stretch.

The giant muscle protein titin is known for its roles in thick-filament formation and the viscoelastic properties of relaxed striated muscles. This protein, also known as connectin, is composed of tandem domains of the fibronectin and immunoglobulin types. Experiments show that the elastic properties come from two sources: from the extension of the polypeptide chain in the PEVK region and the entropy of straightening the molecule.

Immunohistochemistry and polymerase chain reaction were performed on specimens obtained from chick embryonic tissues to study the genetic regulation of myogenesis. The results showed that either signals from the overlying ectoderm or Wnt and Sonic hedgehog signals can induce somitic expression of the paired box transcription factors, Pax-3 and Pax-7, concomitant with the expression of Myf-5 and prior to that of MyoD. The findings also identified the retrovirus gene product Pax-3 as a key regulator of somitic myogenesis.

The ryanodine receptor (RyR) antagonists, ryanodine and daunorubicin, influence the steady-state characteristics and kinetics of intramembrane charge in intact frog skeletal muscle fibers. The antagonists help to retain the total charge across the ON and OFF components of the applied voltage steps. Analyses of the steady-state voltage dependence and pharmacological sensitivities confirm that the RyR antagonists conserve the identity of the q(sub gamma) and q(sub beta) charge species.

Upstream arteriolar diameter increases with increase in metabolic rate of downstream tissue and does not involve the release of epithelium-derived NO or prostaglandins from arteriolar or venular endothelium. Administration of a metabolite stimulates 2, 4-dinitrophenol in the distal region, causing dilation of arterioles followed by increased red cell velocity in the upstream arterioles. This indicates the role of venular-arteriolar diffusion of vasoactive metabolites in the control of upstream arteriolar diameter.

Experiments on horse striated muscles suggest that high plasma creatine kinase (CK) activity levels of more than 10,000 units per liter may cause myolysis. The muscle damage is insignificant for a 60-km test procedure. The amount of CK released during the test is the equivalent of 18.8 plus or minus 4.3 g of striated muscle.

Small RNA within the encoded nucleus of the yeast and mammalian cells are an important subunit of mitochondrial RNA-producing endonuclease (mt DNA) that give rise to primers for mitichondrial DNA duplication. The transcripts of mitochondrial enzymes become abundant in the skeletal muscle prone to motor nerve activation. There is also a need for an upstream stimulation area for expression is cultured skeletal myogenic cells.

Affinity labeling of mu opioid reeceptors in bovine striatal membranes, using (H)-14beta-(Bromoacetamido)-7,8-dihydromorphine (H-3) H2BAM, involves the covalent binding of (H-3) H2BAM to the mu opioid receptor at pH 8.5 and 37 degrees centigrade. It illustrates the pharmacological and molecular properties of the multiple opioid receptors, the magnitude and the specific nature of labeling.

Ryanodine receptors are channel proteins regulating intracellular calcium release in muscle and nonmuscle tissues. These receptors are identified and purified by binding to the plant alkaloid ryanodine, which alters the conductance and gating properties of the channel. Two ryanodine receptor isoforms, alpha and beta, have been identified at the triad junctions of chicken fast twitch skeletal muscle fibers. A third receptor isoform has been localized in avian cardiac muscle. SDS-PAGE, immunological, trypsin digestion and phosphorylation analyses showed that the three isoforms are expressed distinctly from one another in chicken striated muscles.

Investigations are conducted in an animal model of high-grade partial hindlimb ischemia, to assess the degree of interfexene of hydroxyl radical generation and restoration of disrupted reoxygenation in postischemic skeletal muscle, influenced by deferoxamine. Analysis of tissue thiobarbituric acid-reactive substances (TBARS) reveals the extent of lipid peroxidation in skeletal muscle, and membrane dysfunction is determined by measuring resting membrane potential, Em. An increase in muscle TBARS and depolarization of resting Em is observed during ischemia. The functional membrane integrity is influenced by lipid peroxidation during reprefusion phase.

The dependence of the oxygen diffusion coefficient (DOZ) of hamster retractor muscle on temperatures, ranging between 11 to 37 degree celsius, is analyzed by employing a non-steady state technique and measuring the muscle O2 consumption (Vo2), after the steady state is reached. At 11 degree celsius, the value of Do2 is 0.84 plus minus 0.04 times 10 (super -5) sq cm per second. It rises exponentially with an increase in temperature, and at 37 degree celsius, the value of Do2 is estimated to be 2. 41 plus minus 0.19 times 10 (super -5) sq cm per second. At 11 degree celsius, muscle Vo2 is estimated to be 0.35 ml O2 per minute per 100 gm, which increases with the increase in temperature, giving rise to an activation energy of the rate-limiting reaction from the Arrhenius equation.

The mechanochemical interactions between striated muscle cells of jellyfish and the adhering extracellular matrix were studied. Induced DNA replication and proteolytic activity followed by transdifferentiation into RF-amide-positive nerve cells were observed. Moreover, the inhibition of DNA replication and transdifferentiation was suppressed whenever tissues were allowed to form a monolayer of well-stretched cells. The results suggest the mechanochemical interactions play a role in activation and inhibition of DNA replication and transdifferentiation.

Alpha-actinin, a protein from striated muscle, is an endogenous phosphatidylinositol 4,5-biphosphate (PtdInsP2). Moreover, PtdInsP2 controls the F-actin-gelating activity of alpha-actinin by interacting with it. The ability of PtdInsP2 to boost the F-actin-gelating activity of alpha-actinin in smooth muscles to levels normally observed only in striated muscles indicates that PtdInsP2 is requisite for alpha-actinin to maximize its gelating activity.

GLUT-4-containing membranes immunoprecipitated from insulin-stimulated rat skeletal muscle produce the phospholipase D (PLD) product phosphatidic acid. In vitro stimulation of PLD in crude membrane with ammonium sulfate (5 mM) resulted in transfer of GLUT-4 (3.0-fold vs. control) as well as transferrin receptor proteins from large to small membrane structures. The in vitro GLUT-4 transfer could be blocked by neomycin (a PLD inhibitor), and neomycin also reduced insulin-stimulated glucose transport in intact incubated soleus muscles. Furthermore, protein kinase [B.sub.[Beta]] ([PKB.sub.[Beta]]) was found to associate with the GLUT-4 protein and was transferred to small vesicles in response to ammonium sulfate in vitro. Finally, addition of cytosolic proteins, prepared from basal skeletal muscle, and GTP nucleotides to an enriched GLUT-4 membrane fraction resulted in in vitro transfer of GLUT-4 to small membranes (6.8-fold vs. unstimulated control). The cytosol and nucleotide-induced GLUT-4 transfer could be blocked by neomycin and N-ethylmaleimide. In conclusion, we have developed a cell-free assay that demonstrates in vitro GLUT-4 transfer. This transfer may suggest release of GLUT-4-containing vesicles from donor GLUT-4 membranes involving PLD activity and binding of [PKB.sub.[Beta]] to GLUT-4. glucose transport; adenosine diphosphate ribosylation factors; protein kinase [B.sub.[Beta]]; neomycin

In this study, we investigated the hypothesis that prolonged ischemia would impair both sarcoplasmic reticulum (SR) [Ca.sup.2+] uptake and [Ca.sup.2+] release in skeletal muscle. To induce total ischemia (I), a tourniquet was placed around the upper hindlimb in 30 female Sprague-Dawley rats [wt = 256 [+ or -] 6.7 (SE) gl and inflated to 350 mmHg for 4 h. The contralateral limb served as control (C). Immediately after the 4 h of ischemia, mixed gastrocnemius and tibialis anterior muscle was sampled from both limbs, and both crude muscle homogenates and SR vesicles were prepared. In another 10 control animals (CC), muscles were sampled and prepared exactly the same way, but immediately after anesthetization. [Ca.sup.2+] uptake and [Ca.sup.2+] release were measured in vitro with Indo-I on both homogenates and SR vesicles. As hypothesized, submaximal [Ca.sup.2+] uptake was lower (P [is less than] 0.05) in I compared with CC and C, by 25 and 45% in homogenates and SR vesicles, respectively. Silver nitrate ([AgNO.sub.3])-induced [Ca.sup.2+] release, which occurred in two phases (phase I and phase 2), was also altered in I compared with CC and C, in both muscle homogenates and SR vesicles. With ischemia, phase I peak [Ca.sup.2+] release was 26% lower (P [is less than] 0.05) in SR vesicles only. For phase 2, peak [Ca.sup.2+] release was 54 and 24% lower (P [is less than] 0.05) in SR vesicles and homogenates, respectively. These results demonstrate that prolonged skeletal muscle ischemia leads to a reduced SR [Ca.sup.2+] uptake in both homogenates and SR vesicles. The effects of ischemia on SR [Ca.sup.2+] release, however, depend on both the phase examined and the type of tissue preparation. calcium; regulation; metabolism; contractility

The effects of inorganic phosphate ([P.sub.i]) on [Ca.sup.2+] release from the sarcoplasmic reticulum (SR) were studied in mechanically skinned rat skeletal muscle fibers. Application of caffeine or T-tubule depolarization was used to induce [Ca.sup.2+] release from the SR, which was detected using fura 2 fluorescence. Addition of [P.sub.i] (1-40 mM) caused a reversible and concentration-dependent reduction in the caffeine-induced [Ca.sup.2+] transient. This effect was apparent at low [P.sub.i] concentration ([is less than]5 mM), which did not result in detectable precipitation of calcium phosphate within the SR. The inhibitory effect of [P.sub.i] exhibited a marked dependence on free [Mg.sup.2+] concentration. At 0.5 mM free [Mg.sup.2+], 5 mM [P.sub.i] reduced the caffeine-induced transient by 25.1 [+ or -] 4.1% (n = 13). However, at 1.5 mM free [Mg.sup.2+], 5 mM [P.sub.i] reduced the amplitude of caffeine-induced [Ca.sup.2+] transients by 68.9 [+ or -] 3.1% (n = 10). Depolarization-induced SR [Ca.sup.2+] release was similarly affected. These effects of [P.sub.i] may be important in skeletal muscle fatigue, if an inhibitory action of [P.sub.i] on SR [Ca.sup.2+] release is augmented by the rise in cytosolic [Mg.sup.2+] concentration, which accompanies ATP breakdown. calcium; magnesium; sarcoplasmic reticulum; fatigue

Prevention of skeletal muscle insulin resistance by dietary cod protein in high fat-fed rats. Am J Physiol Endocrinol Metab 281: E62-E71, 2001.--In the present study, we tested the hypothesis that fish protein may represent a key constituent of fish with glucoregulatory activity. Three groups of rats were fed a high-fat diet in which the protein source was casein, fish (cod) protein, or soy protein; these groups were compared with a group of chow-fed controls. High-fat feeding led to severe whole body and skeletal muscle insulin resistance in casein- or soy protein-fed rats, as assessed by the euglycemic clamp technique coupled with measurements of 2-deoxy-D-[[sup.3]H]glucose uptake rates by individual tissues. However, feeding cod protein fully prevented the development of insulin resistance in high fat-fed rats. These animals exhibited higher rates of insulin-mediated muscle glucose disposal that were comparable to those of chow-fed rats. The beneficial effects of cod protein occurred without any reductions in body weight gain, adipose tissue accretion, or expression of tumor necrosis factor-a in fat and muscle. Moreover, L6 myocytes exposed to cod protein-derived amino acids showed greater rates of insulin-stimulated glucose uptake compared with cells incubated with casein- or soy protein-derived amino acids. These data demonstrate that feeding cod protein prevents obesity-induced muscle insulin resistance in high fat-fed obese rats at least in part through a direct action of amino acids on insulin-stimulated glucose uptake in skeletal muscle cells. amino acids; high-fat diet; adipose tissue

We tested the hypothesis that denervated muscle fibers account for part of the specific force (s[F.sub.0]) deficit observed in muscles from old adult (OA) mammals. Whole muscle force ([F.sub.0]) was quantified for extensor digitorum longus (EDL) muscles of OA and young adult (YA) rats. EDL muscle s[F.sub.0] was calculated by dividing [F.sub.0] by either total muscle fiber cross-sectional area (CSA) or by innervated fiber CSA. Innervated fiber CSA was estimated from EDL muscle cross sections labeled for neural cell adhesion molecules, whose presence is a marker for muscle fiber denervation. EDL muscles from OA rats contained significantly more denervated fibers than muscles from YA rats (5.6% vs 1.1% of total CSA). When compared with YA muscle, OA muscle demonstrated deficits of 34.1% for [F.sub.0], 28.3% for s[F.sub.0], and 24.9% for s[F.sub.0] calculated by using innervated CSA as the denominator. Denervated muscle fibers accounted for 11.3% of the specific force difference between normal YA and OA skeletal muscle. Other mechanisms in addition to denervation account for the majority of the s[F.sub.0] deficit with aging.

In studies using subtraction cloning to screen for alterations in mRNA expression in skeletal muscle from humans with Type 2 diabetes mellitus and control subjects, one of the most prominent differences was in the mRNA for elongation factor (EF)-1[Alpha]. With Northern blot analysis, EF-1[Alpha] expression was enhanced by 2- to 6-fold in both Types 1 and 2 human diabetics. In contrast, no changes in expression of EF-1[Beta] or -[Gamma] were noted. We observed similar results in animal models of Type I diabetes. EF-1[Alpha] expression, but not EF-1[Beta] or -[Gamma] expression, was also enhanced in streptozotocin-induced diabetic rats, and this effect was reversed by insulin treatment. An increased level of EF-1[Alpha] mRNA was also observed in nonobese diabetic mice. This unbalanced regulation of the expression of the different subunits of EF-1 may contribute to alterations not only in protein synthesis but also in other cellular events observed in the diabetic state. gene expression | insulin | diabetes mellitus

Matrix metalloproteinases (MMP) responsible for degradation of connective tissue are found in most tissues. The MMP are regulated at the levels of transcription, zymogen activation by plasmin or membrane-type- (MT) MMP, and control of enzyme activity by tissue inhibitors of metalloproteinases (TIMP). Whole bovine skeletal muscle showed multiple MMP activities on gelatin zymography and also expressed mRNA encoding MMP-1, -2, -9, -14, and -16, tissue inhibitors of metalloproteinase (TIMP)- 1, -2, and -3 and plasminogen activator and its receptor. Purified intra-muscular fibroblasts and myogenic cell culture derived from satellite cells expressed most or all of these elements. Statistical analysis (n = 35) revealed a strong positive correlation among the mRNA levels of several elements of the MMP system, including MMP-2, MMP-14, TIMP-1, -2, and -3 (r - 0.614 to 0.930, P [is less than] 0.0001). Our results provide an extensive profile of an extracellular proteolytic cascade involving MMP in skeletal muscle and suggest that 1) the activation cascades of muscle MMP may be initiated by both plasmin and membrane-type MMP; 2) a group of genes involved in the same 'arm' of zymogen activation are coexpressed in this tissue; and 3) skeletal muscle cells, in addition to the intramuscular fibroblasts, express an extensive complement of MMP and related proteins. Key Words: Connective Tissue, Fibroblasts, Metalloproteinases, Satellite Cells, Skeletal Muscle

Activity-induced recovery of excitability in [K.sup.+]-depressed rat soleus muscle. Am J Physiol Regulatory Integrative Comp Physiol 280: R48-R55, 2001.--Increased extracellular [K.sup.+] concentration ([[[K.sup.+]].sub.o]) can reduce excitability and force in skeletal muscle. Here we examine the effects of muscle activation on compound muscle action potentials (M waves), resting membrane potential, and contractility in isolated rat soleus muscles. In muscles incubated for 60 rain at 10 mM K+, tetanic force and M wave area decreased to 23 and 24%, respectively, of the control value. Subsequently, short (1.5 s) tetanic stimulations given at 1-min intervals induced recovery of force and M wave area to 81 and 90% of control levels, respectively, within 15 min (P [is less than] 0.001). The recovery of force and M wave was associated with a partial repolarization of the muscle fibers. Experiments with tubocurarine suggest that the force recovery was related to activation of muscle [Na.sup.+]-[K.sup.+] pumps caused by the release of some compound from sensory nerves in response to muscle activity. In conclusion, activity produces marked recovery of excitability in [K.sup.+]-depressed muscle, and this may protect muscles against fatigue caused by increased [[[K.sup.+]].sub.o] during exercise. fatigue; skeletal muscle; contractility; M waves

Cardiac vagal and sympathetic efferent discharges are differentially modified by stretch of skeletal muscle. Am J Physiol Heart Circ Physiol 280: H237-H245, 2001.--We directly measured cardiac vagal efferent nerve activity (CVNA) and cardiac sympathetic efferent nerve activity (CSNA) in cats decerebrated at the level of the precollicular-premammillary body while the hindlimb or the triceps surae muscle was passively stretched. CVNA gradually decreased during passive stretch of the hindlimb, and this decrease was sustained throughout the stretch. CSNA increased at the onset of passive stretch, but this increase was not sustained. CVNA and CSNA responded differentially to graded passive stretches of the triceps surae muscle as well as the hindlimb. The sustained decrease in CVNA but not the initial increase in CSNA became greater depending on muscle length and developed tension. The time course and direction of the cardiac autonomic responses to muscle stretch were not affected by partial sinoaortic denervation, although the magnitude of the CSNA response was augmented. We conclude that the muscle mechanoreflex contributes to differential regulation of cardiac parasympathetic and sympathetic efferent discharges during passive stretch of skeletal muscle irrespective of arterial baroreceptor input. decerebrate cats; partial sinoaortic denervation; muscle mechanoreflex

Leptin administration improves skeletal muscle insulin responsiveness in diet-induced insulin-resistant rats. Am J Physiol Endocrinol Metab 280: E130-E142, 2001.--In addition to suppressing appetite, leptin may also modulate insulin secretion and action. Leptin was administered here to insulin-resistant rats to determine its effects on secretagogue-stimulated insulin release, whole body glucose disposal, and insulin-stimulated skeletal muscle glucose uptake and transport. Male Wistar rats were fed either a normal (Con) or a high-fat (HF) diet for 3 or 6 mo. HF rats were then treated with either vehicle (HF), leptin (HF-Lep, 10 mg [multiplied by] [kg.sup.-1] [multiplied by] day-1 sc), or food restriction (HF-FR) for 12-15 days. Glucose tolerance and skeletal muscle glucose uptake and transport were significantly impaired in HF compared with Con. Whole body glucose tolerance and rates of insulin-stimulated skeletal muscle glucose uptake and transport in HF-Lep were similar to those of Con and greater than those of HF and HF-FR. The insulin secretory response to either glucose or tolbutamide (a pancreatic [Beta]-cell secretagogue) was not significantly diminished in HF-Lep. Total and plasma membrane skeletal muscle GLUT-4 protein concentrations were similar in Con and HF-Lep and greater than those in HF and HF-FR. The findings suggest that chronic leptin administration reversed a high-fat diet-induced insulin-resistant state, without compromising insulin secretion. ob gene product; high-fat diet; glucose tolerance; glucose uptake and transport; GLUT-4 protein

Expression of the [Na.sup.+]/[Ca.sup.2+] exchanger in skeletal muscle. Am J Physiol Cell Physiol 280: C146-C154, 2001.--The expression of the [Na.sup.+]/[Ca.sup.2+] exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform ([Na.sup.+]/[Ca.sup.2+] exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles. sodium/calcium exchanger isoforms; gene expression

Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutation of the gene encoding the cytoskeletal protein dystrophin. Despite a wealth of recent information about the molecular basis of DMD, effective treatment for this disease does not exist because the mechanism by which dystrophin deficiency produces the clinical phenotype is unknown. In both mouse and human skeletal muscle, dystrophin deficiency results in loss of neuronal nitric oxide synthase, which normally is localized to the sarcolemma as part of the dystrophin--glycoprotein complex. Recent studies in mice suggest that skeletal muscle-derived nitric oxide may play a key role in the regulation of blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to [Alpha]-adrenergic receptor activation. Here we report that this protective mechanism is defective in children with DMD, because the vasoconstrictor response (measured as a decrease in muscle oxygenation) to reflex sympathetic activation was not blunted during exercise of the dystrophic muscles. In contrast, this protective mechanism is intact in healthy children and those with polymyositis or limb-girdle muscular dystrophy, muscle diseases that do not result in loss of neuronal nitric oxide synthase. This clinical investigation suggests that unopposed sympathetic vasoconstriction in exercising human skeletal muscle may constitute a heretofore unappreciated vascular mechanism contributing to the pathogenesis of DMD.

Ayada, Kentaro, Makoto Watanabe, and Yasuo Endo. Elevation of histidine decarboxylase activity in skeletal muscles and stomach in mice by stress and exercise. Am J Physiol Regulatory Integrative Comp Physiol 279: R2042-R2047, 2000.--The effects of different types of stress (water bathing, cold, restraint, and prolonged walking) on histidine decarboxylase (HDC) activity in masseter, quadriceps femoris, and pectoralis superficial muscles, and in the stomach were examined in mice. All of these stresses elevated gastric HDC activity. Although water bathing, in which muscle activity was slight, was sufficiently stressful to produce gastric hemorrhage and to increase gastric HDC activity, it produced no detectable elevation of HDC activity in any of the muscles examined. The other stresses all elevated HDC activity in all three muscles. We devised two methods of restraint, one accompanied by mastication and the other not. The former elevated HDC activity in the masseter muscle, but the latter did not. These results suggest that 1) HDC activity in the stomach is an index of responses to stress, 2) the elevation of HDC activity in skeletal muscles during stress is induced partly or wholly by muscle activity and/or muscle tension, and 3) stress itself does not always induce an elevation of HDC activity in skeletal muscles. histamine; masseter muscle; mastication; temporomandibular disorders

Gustafsson, Thomas, Adrian Puntschart, Lennart Kaijser, Eva Jansson, and Carl Johan Sundberg. Exercise-induced expression of angiogenesis-related transcription and growth factors in human skeletal muscle. Am J Physiol 276 (Heart Circ. Physiol. 45): H679-H685, 1999.--mRNA expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and hypoxia-inducible factor (HIF) subunits HIF-1[Alpha], and HIF-1[Beta] in human skeletal muscle was studied during endurance exercise at different degrees of oxygen delivery. Muscle biopsies were taken before and after 45 min of one-legged knee-extension exercise performed under conditions of nonrestricted or restricted blood flow (~15-20% lower) at the same absolute workload. Exercise increased VEGF mRNA expression by 178% and HIF-1[Beta] by 340%, but not HIF-1[Alpha] and FGF-2. No significant differences between the restricted and nonrestricted groups were observed. The exercise-induced increase in VEGF mRNA was correlated to the exercise changes in HIF-1[Alpha] and HIF-1[Beta] mRNA. The changes in VEGF, HIF-1[Alpha], and HIF-1[Beta] mRNAs were correlated to the exercise-induced increase in femoral venous plasma lactate concentration. It is concluded that 1) VEGF but not FGF-2 gene expression is upregulated in human skeletal muscle by a single bout of dynamic exercise and that there is a graded response in VEGF mRNA expression related to the metabolic stress and 2) the increase in VEGF mRNA expression correlates to the changes in both HIF-1[Alpha] and HIF-1[Beta] mRNA.