Showing papers on "Skeletal muscle published in 1989"

Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor.

Abstract: The sequence of 5,037 amino acids composing the ryanodine receptor from rabbit skeletal muscle sarcoplasmic reticulum has been deduced by cloning and sequencing the complementary DNA The predicted structure suggests that the calcium release channel activity resides in the C-terminal region of the receptor molecule, whereas the remaining portion constitutes the 'foot' structure spanning the junctional gap between the sarcoplasmic reticulum and the transverse tubule

Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.

Abstract: Mammalian skeletal muscles consist of three main fibre types, type 1, 2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

Abstract: IN cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel1 represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA2. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested3. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass ∼140,000 (α2-subunit)4–6 associated with the skeletal muscle DHP receptor is co-injected.

A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program.

Abstract: MyoD1 is a nuclear phosphoprotein that is expressed in skeletal muscle in vivo and in certain muscle cell lines in vitro; it has been shown to convert fibroblasts to myoblasts through a mechanism requiring a domain with homology to the myc family of proteins. The BC3H1 muscle cell line expresses skeletal muscle-specific genes upon exposure to mitogen-deficient medium, but does not express MyoD1 at detectable levels. To determine whether BC3H1 cells may express regulatory genes functionally related to MyoD1, a cDNA library prepared from differentiated BC3H1 myocytes, was screened at reduced stringency with the region of the MyoD1 cDNA that shares homology with c-myc. From this screen, a cDNA was identified that encodes a major open reading frame with 72% homology to the myc domain and basic region of MyoD1. The mRNA encoded by this MyoD1-related gene is expressed in skeletal muscle in vivo and in differentiated skeletal myocytes in vitro and is undetectable in cardiac or smooth muscle, nonmuscle tissues, or nonmyogenic cell types. During myogenesis, the MyoD1-related mRNA accumulates several hours prior to other muscle-specific mRNAs and therefore represents an early molecular marker for entry of myoblasts into the differentiation pathway. Transient transfection of 10T1/2 or 3T3 cells with the MyoD1-related cDNA is sufficient to induce myosin heavy-chain expression and to activate a reporter gene under transcriptional control of the muscle creatine kinase 5' enhancer, which functions only in differentiated myocytes. Expression of this cDNA in stably transfected 10T1/2 cells also leads to fusion and muscle-specific gene expression upon exposure to mitogen-deficient medium. Thus, the product of this MyoD1-related gene is sufficient to activate the muscle differentiation program and may substitute for MyoD1 in certain developmental situations. Together, these results suggest the existence of a family of myogenic regulatory genes that share a conserved motif with c-myc.

Identification of MRF4: a new member of the muscle regulatory factor gene family.

Abstract: We have identified a rat cDNA encoding MRF4, a new member of the muscle regulatory factor gene family that includes MyoD1, myogenin, and Myf-5. MRF4 encodes a predicted 27-kD protein that contains a conserved helix-loop-helix motif, which is a common feature of this gene family. Northern analyses indicate that MRF4 is expressed solely in skeletal muscle tissue but is not detected in most embryonic muscle cell lines. Transfection of MRF4 into C3H10T1/2 fibroblasts produces stable myogenic lineages at frequencies that are equal to or greater than those obtained when MyoD1 or myogenin are introduced into these cells. Expression of the MRF4 cDNA leads to expression of the endogenous MyoD1 and myogenin genes, although C3H10T1/2 cells expressing MyoD1 or myogenin cDNAs do not express MRF4. Interestingly, the endogenous MyoD1 and myogenin genes are negatively regulated by serum and by purified growth factors since MRF4-transfected C3H10T1/2 cells activate MyoD1 and myogenin expression only in mitogen-depleted, differentiation-induced muscle cultures. The myofiber-specific expression pattern of MyoD1 and myogenin in these cells suggests that the primary role for this muscle regulatory factor gene family may be in regulating specific terminal differentiation events that are crucial for normal skeletal muscle development.

An autosomal transcript in skeletal muscle with homology to dystrophin.

Abstract: The Duchenne muscular dystrophy (DMD) gene has been localized to chromosome Xp21 and codes for a 14-kilobase (kb) transcript and a protein called dystrophin, of relative molecular mass 427,000. Dystrophin is associated with the cytoplasmic face of muscle fibre membranes and its C-terminal domain is thought to mediate membrane attachment. Although N-terminal and central domain structures share common features with other cytoskeletal components, no significant sequence similarity between the C-terminal region of dystrophin and other previously characterized proteins has been described. Here we report that fragments from the C-terminal domain of the DMD complementary DNA detect a closely related sequence which exhibits nucleic-acid and predicted amino-acid identities with dystrophin of approximately 65 and 80%, respectively. The dystrophin-related sequence identifies a 13-kb transcript in human fetal muscle and maps to chromosome 6. Thus, dystrophin may be a member of a family of functionally related large structural proteins in muscle.

Human variation in skeletal muscle fiber-type proportion and enzyme activities.

Abstract: The purpose of the present study was to describe the extent of the variation in some of the common characteristics of human skeletal muscle. A total of 418 biopsies was obtained from the vastus lateralis muscle of 270 healthy sedentary and 148 physically active individuals of both sexes. The lowest and highest proportion of type I muscle fiber observed were 15 and 85%, respectively. Coefficients of variation (CV) reached approximately 30% for the proportion of types I and IIA fibers and were two times higher for the proportion of type IIB fiber. The smallest and largest mean muscle fiber cross-sectional areas (CSA) were approximately 1,100 microns 2 and 9,500 microns 2, respectively. Mean CSA of the various fiber types exhibited CV of approximately 23%. CV reached 30% for the activity of creatine kinase, ranged between 28 and 41% for the glycolytic enzyme markers, and between 34 and 44% for the aerobic-oxidative enzyme markers. The mean proportion of type I fiber was lower in male than in female muscles, whereas the mean CSA of all fiber types was smaller in female than in male muscles. Levels of glycolytic enzyme markers were higher in male than in female skeletal muscles. However, activities of aerobic-oxidative enzyme markers were similar in males and females. These results reveal the existence of large interindividual variability and gender differences in the most common characteristics of the human skeletal muscle.

Contribution of intrinsic skeletal muscle changes to 31P NMR skeletal muscle metabolic abnormalities in patients with chronic heart failure.

Abstract: Patients with heart failure frequently exhibit abnormal skeletal muscle metabolic responses to exercise, as assessed with 31P NMR. To investigate whether these metabolic abnormalities are due to intrinsic skeletal muscle changes, we performed gastrocnemius muscle biopsies on 22 patients with heart failure (peak VO2, 15.4 +/- 4.7 ml/kg/min; ejection fraction, 20 +/- 7%) and on eight normal subjects. Biopsies were analyzed for fiber type and area, capillarity, citrate synthase, phosphofructokinase, lactate dehydrogenase, and beta-hydroxyacyl CoA dehydrogenase activity. All patients with heart failure also underwent 31P NMR studies of their calf muscle during plantarflexion at three workloads. Muscle pH responses and the relation of the ratio of inorganic phosphate to phosphocreatine (Pi/PCr) to systemic VO2 were then evaluated. Compared with normal subjects, patients with heart failure exhibited a shift in fiber distribution with increased percentage of the fast twitch, glycolytic, easily fatigable type IIb fibers (normal subjects, 22.7 +/- 10.1; heart failure, 33.1 +/- 11.1%; p less than 0.05), atrophy of type IIa (normal subjects, 5,477 +/- 1,109; heart failure, 4,239 +/- 1,247 microns 2; p less than 0.05) and type IIb fibers (normal subjects, 5,957 +/- 1,388; heart failure, 4,144 +/- 945 microns 2; p less than 0.01), and decreased activity of beta-hydroxyacyl CoA dehydrogenase (normal subjects, 5.17 +/- 1.44; heart failure, 3.67 +/- 1.68 mol/kg protein/hr; p less than 0.05). No significant linear correlation could be identified between the slope of the Pi/PCr to VO2 relation and muscle histochemistry or enzyme activities. Similarly, no linear relation was found between intracellular pH at peak exercise and any muscle variable.(ABSTRACT TRUNCATED AT 250 WORDS)

Infusion of tumor necrosis factor/cachectin promotes muscle catabolism in the rat. A synergistic effect with interleukin 1.

Abstract: To improve our understanding of the metabolic role of cytokines in protein wasting, we estimated the rates of protein synthesis and degradation in muscle and liver tissues in intact rats treated with several doses of recombinant IL 1 and/or tumor necrosis factor (TNF)/cachectin. Protein breakdown in muscle and liver were derived in vivo from the relationship between [14C]leucine distribution and tissue dilution in reference to circulating leucine. Synthesis was derived from the relationship between [14C]leucine appearance in the protein-bound and free-tissue leucine pools. To specifically relate changes in leucine tracer metabolism to protein dynamics, we separately measured the effect of these cytokines on blood flow to different tissues. The increase in dilution of the tissue-free [14C]leucine by TNF and TNF/IL 1 mixture, but not by IL 1 alone, could not be explained by a hemodynamic effect of these cytokines. Rather, this finding indicated that muscle proteolysis is enhanced by TNF and synergistically augmented by the addition of IL 1. Compatible with these data was the finding that more prolonged infusions of recombinant TNF/cachectin and the combination with IL 1 increased urinary nitrogen excretion. Changes in [14C]leucine dilution in the liver were less pronounced than those in skeletal muscle and consistent with net anabolic effect of TNF on liver protein. We conclude that rats exposed systemically to sublethal doses of TNF respond with increasing muscle and decreasing liver proteolysis, similar to that observed in inflammation and in cancer.

Molecular cloning of a putative tetrodotoxin-resistant rat heart Na+ channel isoform.

Abstract: Voltage-gated Na+ channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na+ channels are blocked by 1-10 microM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity [3H]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire alpha subunit of a cardiac-specific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles.

Effect of exercise on insulin action in human skeletal muscle

Abstract: The effect of 1 h of dynamic one-legged exercise on insulin action in human muscle was studied in 6 healthy young men. Four hours after one-legged knee extensions, a three-step sequential euglycemic hyperinsulinemic clamp combined with arterial and bilateral femoral vein catheterization was performed. Increased insulin action on glucose uptake was found in the exercised compared with the rested thigh at mean plasma insulin concentrations of 23, 40, and 410 microU/ml. Furthermore, prior contractions directed glucose uptake toward glycogen synthesis and increased insulin effects on thigh O2 consumption and at some insulin concentrations on potassium exchange. In contrast, no change in insulin effects on limb exchange of free fatty acids, glycerol, alanine or tyrosine were found after exercise. Glycogen concentration in rested vastus lateralis muscle did not increase measurably during the clamp even though indirect estimates indicated net glycogen synthesis. In contrast, in exercised muscle estimated and biopsy-verified increases in muscle glycogen concentration agreed. Local contraction-induced increases in insulin sensitivity and responsiveness play an important role in postexercise recovery of human skeletal muscle.

Classical conditioning in rabbits using pontine nucleus stimulation as a conditioned stimulus and inferior olive stimulation as an unconditioned stimulus.

Abstract: Classical conditioning of skeletal muscle responses was accomplished by pairing microstimulation of the pontine nuclei as a conditioned stimulus (CS) with microstimulation of the dorsal accessory olive as an unconditioned stimulus (US). A conditioned response identical in form to the behavioral response elicited by the olivary stimulation was established when the CS was forward paired with the US, and behavioral extinction occurred with CS-alone presentations or unpaired CS-US presentations. Conditioned responding could not be established or maintained when the CS and US were simultaneously presented or when the US preceded the CS (i.e., backward paired). Complete lesions of the interpositus nucleus abolished both conditioned and unconditioned responses. These findings support the idea that plasticity associated with classical conditioning of skeletal muscle responses occurs in regions of the cerebellum that receive convergent CS and US input.

Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle.

Abstract: 1. Maximal calcium-activated force (Fmax) and calcium sensitivity were markedly decreased in detergent-skinned fibres from skeletal and cardiac muscle by solutions that mimicked the total milieu changes associated with fatigue and hypoxia. Further experiments determined the relative contribution of each of the individual changes in milieu. 2. Both Ca2+ sensitivity and Fmax of skeletal and cardiac fibres were decreased with increased [H+] or inorganic phosphate (Pi). These effects were greater in cardiac muscle. 3. Decreasing MgATP over the range observed with fatigue and hypoxia (6.8-4.7 mM) had no effect on Fmax or Ca2+ sensitivity of either muscle type. 4. Decreasing phosphocreatine (PCr: 15-1 mM) increased Fmax but had little effect on Ca2+ sensitivity in both muscle types. In cardiac fibres, the effect on Fmax could be mimicked by inhibition of endogenous creatine kinase. 5. ADP (0.7 mM) increased Fmax and Ca2+ sensitivity, while AMP (0.06 mM) slightly increased Fmax but had no effect on Ca2+ sensitivity of either skeletal or cardiac fibres. 6. Creatine (25 mM) had no significant effect on either Ca2+ sensitivity or Fmax of skeletal and cardiac muscle fibres. At higher levels (50 mM), however, creatine depressed Fmax and slightly altered Ca2+ sensitivity. 7. Thiophosphorylation of myosin P light chains (phosphorylatable light chains of myosin) in rabbit psoas fibres had no effect on Ca2+ sensitivity, yet slightly but significantly increased Fmax under fatigue conditions. 8. Reducing the affinity for ATP hydrolysis (by adding ADP, AMP and creatine) over the range calculated for fatigue/hypoxia (60-45 kJ/mol) produced the enhancement in Fmax expected from added ADP and AMP in cardiac but not skeletal muscle, indicating that changes in affinity influence Fmax of skeletal muscle. Reducing affinity produced little change in Ca2+ sensitivity of skeletal muscle. In contrast, the change produced in cardiac muscle was greater than that expected from addition of ADP and AMP; i.e. decreasing affinity increases calcium sensitivity of the heart. 9. Simple summation of all significant changes expected from each constituent altered by fatigue/hypoxia adequately predicted the observed changes in Fmax and Ca2+ sensitivity in both cardiac and skeletal muscle fibres with but one exception (the change in Ca2+ sensitivity of skeletal muscle at pH 7 was slightly overestimated).

Induction of calcium currents by the expression of the alpha 1-subunit of the dihydropyridine receptor from skeletal muscle.

Abstract: The dihydropyridine (DHP) receptor purified from skeletal muscle comprises five protein subunits (alpha 1, alpha 2, beta, gamma and delta) and produces Ca2+ currents that are blocked by DHPs. Cloning of the alpha 1- and alpha 2-subunits, the former affinity-labelled by DHP, has shown that the alpha 1-subunit is expressed in skeletal muscle alone, whereas the alpha 2- and delta- subunits are also expressed in other tissues. Although the transient expression of the alpha 1-subunit in myoblasts from dysgenic mice (but not in oocytes) has been demonstrated, the use of these expression systems to determine the function of the alpha 1- subunit is complicated by the presence of endogenous Ca2+ currents, which may reflect the constitutive expression of proteins similar to the alpha 2-, beta-, gamma- and/or delta-subunits. We therefore selected a cell line which has no Ca2+ currents or alpha 2- subunit, and probably no delta-subunit for stable transformation with complementary DNA of the alpha 1- subunit. The transformed cells express DHP-sensitive, voltage-gated Ca2+ channels, indicating that the minimum structure of these channels is at most an alpha 1 beta gamma complex and possibly an alpha 1- subunit alone.

Multiple actions of beta-adrenergic agonists on skeletal muscle and adipose tissue.

Abstract: β-adrenergic agonists (β-agonits) an potent growth promoters in many species of animals. This class of compounds produces a dramatic increase in skeletal muscle mass and a large reduction in body fat content. The physiological findings detailed below may provide the initial basis for investigations to understand these processes botter

Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum

Abstract: THE calcium channel responsible for the release of Ca2+ from the sarcoplasmic reticulum of skeletal muscle during excitation–contraction coupling has recently been identified and purified1–4,21. The isolated calcium channel has been identified morphologically with the 'foot' structures1,2 which are associated with the junctional face membrane of the terminal cisternae of sarcoplasmic reticulum. In situ, the foot structure extends across the gap of the triad junction from the terminal cisternae of the reticulum to the trans-verse tubule5. We describe here the three-dimensional architecture (3.7 nm resolution) of the calcium channel/foot structure from fast-twitch rabbit skeletal muscle, which we determined from electron micrographs of isolated, non-crystalline structures that had been tilted in the electron microscope. The reconstruction reveals two different faces and an internal structure in which stain accumulates at several interconnected locations, which could empty into the junctional gap of the triad junction. The detailed architecture of the channel complex is relevant to understanding both the physical path followed by calcium ions during excitation–contraction coupling and the association of the terminal cisternae and the transverse tubules in the triad junction.

Noninvasive detection of skeletal muscle underperfusion with near-infrared spectroscopy in patients with heart failure.

Abstract: The present study was undertaken to determine whether near-infrared spectroscopy can be used to noninvasively assess skeletal muscle oxygenation in patients with heart failure. The difference between light absorption at 760 and 800 nm was used to assess hemoglobin-myoglobin oxygenation. Initial studies conducted in isolated canine gracilis muscle demonstrated that 760-800-nm absorption correlated closely (r = -0.97 +/- 0.01) with venous hemoglobin O2 saturation when the muscle was stimulated to contract at 0.25-5.0 Hz. In normal subjects (n = 6) and patients with heart failure (n = 8), 760-800-nm absorption changes from the vastus lateralis muscle were monitored at rest, during progressive maximal bicycle exercise, and during thigh cuff inflation to suprasystolic pressure, an intervention designed to assess minimal hemoglobin-myoglobin oxygenation. Absorption changes were expressed relative to the full physiologic range noted from rest to thigh cuff inflation. During exercise, normal subjects exhibited an initial increase in hemoglobin-myoglobin oxygenation followed by a progressive decrease in oxygenation to 27 +/- 13% of the physiologic range at the peak exercise workload of 140 +/- 9 W. In contrast, patients exhibited an initial decrease in hemoglobin-myoglobin oxygenation with the first workload, followed by a progressive further decrease to 26 +/- 13% of the physiologic range at a peak exercise workload of 60 +/- 8 W, less than half the peak workload noted in the normal subjects. At all exercise loads, hemoglobin-myoglobin oxygenation was significantly less in the patients than in the normal subjects. These data suggest that near-infrared spectroscopy can detect impaired skeletal muscle O2 delivery in patients with heart failure. This technique could provide a valuable method of assessing muscle O2 delivery in patients, particularly before and after therapeutic interventions.

Cachectin/TNF or IL-1 alpha induces cachexia with redistribution of body proteins

Abstract: Macrophage secretory products are suspected to participate in the severe lean tissue wasting related to chronic illness. The protein metabolic effects of chronic, 7-day cachectin/tumor necrosis factor (cachectin) or interleukin 1 alpha (IL-1 alpha) administration in vivo were studied in male Wistar rats that were 1) freely fed, 2) pair fed, 3) total protein and calorie starved, 4) twice daily lipopolysaccharide (LPS) administered, 5) twice daily cachectin administered, and 6) twice daily IL-1 alpha administered. LPS, cachectin, or IL-1 alpha administration produced anorexia; weight loss in these groups was comparable to respective pair-fed animals. However, LPS, cachectin, or IL-1 alpha accelerated peripheral protein wasting while preserving liver protein content, unlike the pattern in the pair-fed or starved animals in which loss of liver proteins and relative preservation of skeletal muscle protein were observed. The decrease in skeletal muscle protein content in LPS- or cytokine-treated animals was associated with coordinate decreases in muscle mRNA levels for the myofibrillar proteins myosin heavy chain, myosin light chain, actin, and in the 18S and 28S subunits of ribosomal RNA. We conclude that chronic exposure to the cytokines, IL-1 alpha or cachectin, can simulate those body and muscle protein changes seen in experimental LPS administration or chronic disease and markedly differ from the pattern of protein redistribution due to caloric restriction.

Fibroblast growth factor in the extracellular matrix of dystrophic (mdx) mouse muscle.

Abstract: Polyclonal antibody F547 reacts with a bovine basic fibroblast growth factor (bFGF) and a human recombinant bFGF, but not with bovine acidic fibroblast growth factor. This antibody localized bFGF in the extracellular matrix of mouse skeletal muscle, primarily in the fiber endomysium, which includes the heparin-containing basal lamina. In mdx mouse muscle, which displays persistent regeneration, FGF levels in the extracellular matrix are higher than those in controls. Overabundance of matrix FGF in mdx muscles may be related to an increase in both satellite cell and regenerative activity in the dystrophic muscle and may help explain the benign phenotype of mdx animals compared with the genetically identical human Duchenne muscular dystrophy.

Thermal acclimation induces adaptive changes in subcellular structure of fish skeletal muscle.

Abstract: Stereological analyses of electron micrographs were used to quantify physiologically important ultrastructures of slow-twitch oxidative (red) and fast-twitch glycolytic (white) muscle fibers from striped bass (Morone saxatilis) acclimated to 25 and 5 degrees C. The fraction of cell volume occupied by the mitochondria [volume density, Vv (mit,f)] of red fibers increases from 0.286 +/- 0.018 to 0.448 +/- 0.024 between 25 and 5 degrees C; Vv (mit,f) of white fibers increased from 0.027 +/- 0.003 and 0.040 +/- 0.004 at 25 and 5 degrees C, respectively. Because of a concomitant increase in the mass of oxidative muscle, acclimation from 25 to 5 degrees C results in an increase in total mitochondrial volume per 100 g body wt from 2.58 to 6.73 cm3 in oxidative muscle and from 2.46 to 3.40 cm3 in fast glycolytic muscle. Mitochondria of red fibers are in more clustered arrays after cold acclimation. Size and cristae surface densities of individual mitochondria are not affected markedly by acclimation, suggesting true cold-induced proliferation rather than enlargement of organelles. Harmonic means of intermitochondrial spacing in red fibers decreases from 2.64 to 1.43 micron between 25 and 5 degrees C. This reduces diffusion path lengths between sarcoplasmic and mitochondrial compartments proportionately, compensating for decreases in diffusivity of aqueous solutes. Intracellular lipid droplets of red fibers markedly increase in volume density from 0.006 +/- 0.003 at 25 degrees C to 0.079 +/- 0.014 at 5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of partial neuromuscular blockade on sympathetic nerve responses to static exercise in humans.

Abstract: We used intraneural recordings of sympathetic nerve activity in conscious humans to determine if central command increases sympathetic discharge to resting skeletal muscle during static exercise. In nine healthy subjects, we measured arterial pressure, heart rate, and muscle sympathetic nerve activity with microelectrodes in the peroneal nerve of the resting leg during 1) static handgrip at 15% and 30% maximal voluntary contraction and 2) attempted handgrip during partial neuromuscular blockade produced by systemic administration of tubocurarine chloride (0.075 mg/kg i.v.). During curare, subjects reported that they used near-maximal motor effort to attempt a sustained handgrip contraction, but they generated almost no force. Without sustained contraction, the intent to exercise alone, that is, central command, caused statistically significant (p less than 0.05) increases in muscle sympathetic nerve activity as well as in arterial pressure and heart rate. However, the increases in muscle sympathetic nerve activity (+ 56 +/- 16% over control) and in mean arterial pressure (+ 12 +/- 2 mm Hg) during attempted handgrip were much smaller (p less than 0.05) than the sympathetic nerve response (+ 217 +/- 37% over control) and pressor response (+ 25 +/- 3 mm Hg) during an actual static handgrip at 30% maximal voluntary contraction. In contrast, heart rate increased as much during the attempted contraction (+ 18 +/- 2 beats/min) as during the actual contraction at 30% maximal voluntary contraction (+ 16 +/- 4 beats/min). In 11 additional subjects, the heart rate responses during curare were greatly attenuated (p less than 0.05) by atropine but were not significantly affected by propranolol.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical influences on long-lasting human muscle fatigue and delayed-onset pain.

Abstract: 1. The influence of three mechanical factors, force, muscle length and passive lengthening, on long-lasting changes in voluntary force generation, the force:frequency relationship and the development of tenderness has been studied in healthy human skeletal muscle. The elbow flexors were used in all studies. The effect of muscle length was also investigated in the quadriceps and adductor pollicis muscles. Eighty maximal voluntary contractions (MVCs) were performed: one contraction, lasting approximately 2 s, every 15 s. The MVC and force:frequency relationships were measured before and immediately after the exercise and, together with an assessment of tenderness, at 24 h intervals thereafter. 2. In a series of experiments designed to investigate the effects of force, eccentric (lengthening) contractions were found to cause greater fatique and delayed-onset muscle pain than either isometric or concentric (shortening) contractions. There were, however, no substantial differences between the effects of isometric and concentric contractions. Changes in MVC took 24-48 h to return to normal while the low-frequency fatigue required 3-4 days to recover. 3. Passive lengthening with a comparable number of movements over the full range had no effect on the force generation of the muscle, nor did it cause any muscle pain. 4. In the series of experiments designed to investigate the effects of length, isometric MVCs were performed at either short or long length and the muscles subsequently tested at an intermediate length. The contractions at long length resulted in greater low-frequency fatigue and pain, despite the fact that they generated less force than those at the short length. 5. The results demonstrate that there is no simple relationship between the force generated during exercise and the development of long-lasting muscle fatigue and pain. Furthermore, there is a length-dependent component in the generation of low-frequency fatigue and muscle pain.

Duchenne muscular dystrophy gene product is not identical in muscle and brain

Abstract: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting in progressive degeneration of the muscle. It affects about 1 in 3,500 male children. Becker's muscular dystrophy is a less severe disease allelic to DMD. Some 30% of DMD patients suffer from various degrees of mental retardation. The giant DMD gene spans about 2,000 kilobases and codes for a 14-kilobase messenger RNA and a protein of molecular weight 427,000. DMD mRNA is most abundant in skeletal and cardiac muscle and less so in smooth muscle. We reported that the expression of the gene is developmentally regulated during the differentiation of primary muscle cultures and in myogenic cell lines in a way similar to the expression of muscle-specific genes such as myosin light chain 2 and skeletal muscle actin. Similar results have been obtained with human primary myogenic cells. Significant levels of DMD mRNA are found in brain tissue. Here we show that the transcript of the DMD gene and the amino terminal of the encoded protein differ in brain and muscle. The 5' ends of these mRNA species are derived from different exons. The results suggest that the two mRNA types are transcribed from different promoters.

Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

Abstract: Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.