Localization of Myf-5, MRF4 and alpha cardiac actin mRNAs in regenerating Xenopus skeletal muscle

Dendrotoxin I (DTX-I) is a 60-residue peptide from the venom of the black mamba snake Dendroaspis polylepis, which binds to neuronal K+ channels. The structure reported previously for DTX-I was synthesized for the first time by a solution procedure. The synthetic product was confirmed to have the correct primary and disulfide structure determined by peptide mapping, sequence analysis and mass measurements. Comparison of synthetic DTX-I with the natural one by high-performance liquid chromatography and capillary zone electrophoresis, as well as by sequence analysis, revealed that the Asn residue at position 12 in the synthetic peptide was Asp in the natural product. Synthesis of DTX-I with Asp at position 12 gave a peptide identical with the natural product in all aspects. NMR analysis of synthetic [Asn12]- and [Asp12]-DTX-I also supported our findings that the Asn residue at position 12 in the DTX-I molecule should be revised as Asp. [Asn12]- and [Asp12]-DTX-I had very similar binding affinities when tested against radiolabeled dendrotoxin binding to rat brain synaptosomal membranes.     

Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus

Three different null alleles of the myogenic bHLH gene MRF4/herculin/Myf-6 were created recently. The three alleles were similar in design but were surprisingly different in the intensity of their phenotypes, which ranged from complete viability of homozygotes to complete lethality. One possible explanation for these differences is that each mutation altered expression from the nearby Myf-5 gene to a different extent. This possibility was first raised by the observation that the most severe MRF4 knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of the Myf-5 null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viability correlate with the intensity of rib skeletal defects, and the most extreme version of this rib defect is the hallmark phenotype of Myf-5 null animals. In the present study we tested this hypothesis for the two milder MRF4 alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakest MRF4 knockout allele on one chromosome 10 and a Myf-5 knockout allele on the other chromosome, we found that both of these MRF4 alleles apparently downregulate Myf-5 expression by a cis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viable MRF4 allele, together with intensified rib defects for both MRF4 alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis for cis interactions at the MRF4/Myf-5 locus was further examined by fusing a DNA segment containing the entire MRF4 structural gene, including all sequences deleted in the three MRF knockout alleles, with a basal promoter and a lacZ reporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA. cis-acting interactions between Myf-5 and MRF4 may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos.     

The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells

The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle-dependent regulation, thus establishing a correlation between the cell cycle-specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.     

MyoD and the specification of muscle and non-muscle fates during postembryonic development of the C. elegans mesoderm

Basic-helix-loop helix factors of the myoD/myf5/ myogenin/MRF4 family have been implicated in acquisition and elaboration of muscle cell fates. Here we describe both myogenic and non-myogenic roles for the Caenorhabditis elegans member of this family (CeMyoD) in postembryonic mesodermal patterning. The postembryonic mesodermal lineage in C. elegans provides a paradigm for many of the issues in mesodermal fate specification: a single mesoblast ('M') divides to generate 14 striated muscles, 16 non-striated muscles, and two non-muscle cells. To study CeMyoD function in the M lineage, we needed to circumvent an embryonic requirement for the protein. Two approaches were used: (1) isolation of mutants that decrease CeMyoD levels while retaining viability, and (2) analysis of genetic mosaics that had lost CeMyoD in the M lineage. With either manipulation, we observed a series of cell-fate transformations affecting a subset of both striated muscles and non-muscle cells. In place of these normal fates, the affected lineages produced a number of myoblast-like cells that initially failed to differentiate, instead swelling to acquire a resemblance to sex myoblasts (M-lineage-derived precursors to non-striated uterine and vulval muscles). Like normal sex myoblasts, the ectopic myoblast-like cells were capable of migration and proliferation followed by differentiation of progeny cells into vulval and uterine muscle. Our results demonstrate a cell-intrinsic contribution of CeMyoD to specification of both non-muscle and muscle fates.     

Distinct signaling pathways regulate transformation and inhibition of skeletal muscle differentiation by oncogenic Ras

Expression of oncogenic Ras in 23A2 skeletal myoblasts is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype, but the signaling pathways activated by oncogenic Ras in these cells and their respective contribution to each phenotype have not been explored. In this study, we investigated MAP kinase activity in control 23A2 myoblasts and in 23A2 myoblasts rendered differentiation-defective by the stable expression of an oncogenic (G12V)Ha-ras gene (Ras9 cells). The MAP kinase immunoprecipitated from Ras9 cells was 30-40% more active than that from control 23A2 cells. To establish if this elevated MAP kinase activity is essential to the maintenance of the oncogenic Ras-induced phenotype, we utilized the selective MAP kinase kinase 1 (MEK1) inhibitor, PD 098059. PD 098059 decreased the MAP kinase activity in Ras9 cells to the level found in 23A2 cells. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. PD 098059 reverted the transformed morphology of Ras9 cells but did not restore the ability of these cells to express the muscle-specific myosin heavy chain gene or to form muscle fibers. Treatment with PD 098059 also did not affect the ability of oncogenic Ha-Ras to establish a non-myogenic phenotype in C3H10T1/2 cells co-expressing MyoD. These results demonstrate that the activation of MAP kinase is necessary for the transformed morphology of Ras9 cells but is not required by oncogenic Ras to establish or to maintain a differentiation-defective phenotype. While these data do not rule out the possibility that constitutive signaling by MEK1 or MAP kinase could inhibit myoblast differentiation, they clearly demonstrate that other pathways activated by oncogenic Ras are sufficient to inhibit differentiation.     

Myosin heavy chain isoforms and dynamic contractile properties: skeletal versus smooth muscle

Torsades de pointes is a potentially life-threatening form of polymorphic ventricular tachyarrhythmia typically seen in the presence of repolarization-prolonging agents. We investigated this particular form of tachyarrhythmia in the isolated, perfused rabbit heart. The experimental model was designed to reproduce conditions that are clinically known to be associated with an increased propensity to the development of torsades de pointes. The class III agent clofilium (1 microM) and d,l-sotalol (10 microM), as well as the antibiotic erythromycin (30-150 microM) were infused in the presence of either normal (5.88 mM) or low (1.5 mM) potassium concentration in sinus-driven or atrioventricular (AV)-blocked hearts. Ventricular tachyarrhythmias spontaneously emerged in the clofilium-, d,l-sotalol-, and erythromycin-treated AV-blocked hearts. The episodes showed typical features of torsades de pointes found in humans. They developed within 4-12 min after the onset of infusion, were normally nonsustained, and only rarely degenerated into ventricular fibrillation. Electrical stimulation at cycle lengths <600 ms and perfusion with MgSO4 suppressed arrhythmic activity. In the d,l-sotalol- and erythromycin-treated hearts, torsades de pointes occurred only in the presence of hypokalemia and bradycardia, whereas, in the presence of clofilium, bradycardia alone caused torsades de pointes. Monophasic action-potential recordings demonstrated early afterdepolarizations in endocardial and epicardial recordings. Thus the isolated AV-blocked rabbit heart represents a model for studying drug-related torsades de pointes and its mechanism.     

Characterization of skeletal muscle actin labeled with the triplet probe erythrosin-5-iodoacetamide

We have labeled rabbit skeletal muscle actin with the triplet probe erythrosin-5-iodoacetamide and characterized the labeled protein. Labeling decreased the critical concentration and lowered the intrinsic viscosity of F-actin filaments; labeled filaments were motile in an in vitro motility assay but were less effective than unlabeled F-actin in activating myosin S1 ATPase activity. In unpolymerized globular actin (G-actin), both the prompt and delayed luminescence were red-shifted from the spectra of the free dye in solution and the fluorescence anisotropy of the label was high (0.356); filament formation red shifted all excitation and emission spectra and increased the fluorescence anisotropy to 0.370. The erythrosin phosphorescence decay was at least biexponential in G-actin with an average lifetime of 99 microseconds while in F-actin the decay was approximately monoexponential with a lifetime of 278 microseconds. These results suggest that the erythrosin dye was bound at the interface between two actin monomers along the two-start helix. The steady-state phosphorescence anisotropy of F-actin was 0.087 at 20 degrees C and the anisotropy increased to approximately 0.16 in immobilized filaments. The phosphorescence anisotropy was also sensitive to binding the physiological ligands phalloidin, cytochalasin B and tropomyosin. This study lays a firm foundation for the use of this triplet probe to study the large-scale molecular dynamics of F-actin.     

Hematomas and limb skeletal malformations in chicken embryos following exposure to 5-fluoro-2-deoxyuridine

Despite considerable attention to community integration and related topics in the past decades, a clear definition of community integration continues to elude researchers and service providers. Common to most discussions of the topic, however, are three ideas: that integration involves relationships with others, independence in one's living situation and activities to fill one's time. The present study sought to expand this conceptualization of community integration by asking people with brain injuries for their own perspectives on community integration. This qualitative study resulted in a definition of community integration consisting of nine indicators: orientation, acceptance, conformity, close and diffuse relationships, living situation, independence, productivity and leisure. These indicators were empirically derived from the text of 116 interviews with people with moderate-severe brain injuries living in the community. Eighteen adults living in supported living programmes were followed for 1 year, to track their evolving definition of integration and the factors they felt were related to integration. The study also showed a general trend toward more positive evaluation over the year, and revealed that positive evaluation was frequently related to meeting new people and freedom from staff supervision. These findings are interpreted in the light of recommendations for community programmes.     

Genetic analysis of alpha 4 integrin functions in the development of mouse skeletal muscle

It has been suggested, on the basis of immunolocalization studies in vivo and antibody blocking experiments in vitro, that alpha 4 integrins interacting with vascular cell adhesion molecule 1 (VCAM-1) are involved in myogenesis and skeletal muscle development. To test this proposal, we generated embryonic stem (ES) cells homozygous null for the gene encoding the alpha 4 subunit and used them to generate chimeric mice. These chimeric mice showed high contributions of alpha 4-null cells in many tissues, including skeletal muscle, and muscles lacking any detectable (< 2%) alpha 4-positive cells did not reveal any gross morphological abnormalities. Furthermore, assays for in vitro myogenesis using either pure cultures of alpha 4-null myoblasts derived from the chimeras or alpha 4-null ES cells showed conclusively that alpha 4 integrins are not essential for muscle cell fusion and differentiation. Taking these results together, we conclude that alpha 4 integrins appear not to play essential roles in normal skeletal muscle development.     

mRNA for cardiac calcium channel is expressed during development of skeletal muscle

During the early development of skeletal muscle, cardiac isotypes of several contractile proteins are known to be transiently expressed. We report here that skeletal muscle developing in vivo, as well as primary cultures derived from skeletal muscle, express mRNA encoding the cardiac dihydropyridine-sensitive calcium channel. The mRNA is detectable at high concentration at the earliest stage tested in vivo and diminishes rapidly in concentration as myofibers mature. The concentration of the cardiac calcium channel mRNA also diminishes during the in vivo development of skeletal muscle in a genetically paralyzed mouse (mdg), indicating that muscle contractile activity is not necessary for the down-regulation. In contrast, mRNA for the skeletal muscle-specific calcium channel accumulates gradually in developing skeletal muscle. A similar temporal pattern of expression is also seen in primary cultures of skeletal myotubes. These results raise the question of whether the cardiac calcium channel may be functionally important during the early development of skeletal myofibers.     

Distinct dendritic cell subsets differentially regulate the class of immune response in vivo

Dendritic cells (DCs) are unique in their ability to stimulate T cells and initiate adaptive immunity. Injection of mice with the cytokine Flt3-ligand (FL) dramatically expands mature lymphoid and myeloid-related DC subsets. In contrast, injection of a polyethylene glycol-modified form of granulocyte/macrophage colony-stimulating factor (GM-CSF) into mice only expands the myeloid-related DC subset. These DC subsets differ in the cytokine profiles they induce in T cells in vivo. The lymphoid-related subset induces high levels of the Th1 cytokines interferon gamma and interleukin (IL)-2 but little or no Th2 cytokines. In contrast, the myeloid-related subset induces large amounts of the Th2 cytokines IL-4 and IL-10, in addition to interferon gamma and IL-2. FL- or GM-CSF-treated mice injected with soluble ovalbumin display dramatic increases in antigen-specific antibody titers, but the isotype profiles seem critically dependent on the cytokine used. Although FL treatment induces up to a 10, 000-fold increase in ovalbumin-specific IgG2a and a more modest increase in IgG1 titers, GM-CSF treatment favors a predominantly IgG1 response with little increase in IgG2a levels. These data suggest that distinct DC subsets have strikingly different influences on the type of immune response generated in vivo and may thus be targets for pharmacological intervention.     

Relative importance of growth hormone and sex steroids for the growth at puberty of trunk length, limb length, and muscle width in growth hormone-deficient children

We have followed the growth of stature, sitting height, skinfolds, muscle widths measured radiologically, and skeletal maturity in growth hormone-deficient patients in whom hGH was given and withheld in alternating three-month periods throughout puberty (referred to as "off-hGH" and "on-hGH" periods). Six boys and four girls had true isolated GH deficiency and developed puberty spontaneously. Two boys had gonadotrophin deficiency plus GH deficiency, and five boys had multiple deficiencies; in these boys the signs of puberty were induced by hormone treatment. Boys with true isolated deficiency grew about two-thirds as much in height in the off-hGH periods as in the on-hGH periods; their total gain in height during the adolescent spurt would have been about 20 cm, instead of 30 cm, if hGH had been discontinued at the beginning of puberty. The effect of hGH was entirely on growth in leg-length, however, which virtually ceased during the off-hGH periods. Growth in sitting height altered little when hGH was withdrawn. Growth in limb muscles, however, was GH dependent throughout puberty; during the majority of periods when hGH was withheld, muscle was actually lost; this occurred in the boys who were receiving large doses of testosterone as well as in those producing their own normal amounts. Subcutaneous fat diminished when hGH was given and increased when it was withdrawn; this occurred independently of administration of testosterone. There was little evidence that growth of pubic and axillary hair progressed faster during on-hGH periods, except perhaps in patients with multiple deficiencies. There was some evidence, however, that bone age progressed less rapidly during on-hGH periods than during off-hGH periods in the patients with isolated deficiency. The results in the girls agreed with those in boys so far as stature was concerned, but the relationship with sitting height and leg length appeared to be different; the reasons for this are discussed. We conclude that all children with GH deficiency should continue on treatment with hGH throughout puberty, ideally until growth ceases.     

Cross-sectional study of the isokinetic muscle trunk strength among school children

Our surveys have shown lifetime prevalence of L.BP. over 30% among schoolchildren. The purpose of this study was to evaluate the relationship between back and isokinetic trunk strength, anthropometric parameters, and sports activities. One hundred and seventeen healthy children aged 10-16 years were included. All these volunteers had semi-structured interview, anthropometric and dynamic strength measurements. Lifetime prevalence of back pain was 44.5% and point prevalence was 13%. In this cross-sectional study, anthropometric and strength profiles were significantly related to age and gender. Non specific low back pain was not correlated to trunk muscle strength and/or sports activities.     

Role of thyroid hormones in early postnatal development of skeletal muscle and its implications for undernutrition

Energy intake profoundly influences many endocrine axes which in turn play a central role in development. The specific influence of a short period of mild hypothyroidism, similar to that induced by undernutrition, in regulating muscle development has been assessed in a large mammal during early postnatal life. Hypothyroidism was induced by providing methimazole and iopanoic acid in the feed of piglets between 4 and 14 d of age, and controls were pair-fed to the energy intake of their hypothyroid littermates. Thyroid status was evaluated, and myofibre differentiation and cation pump concentrations were then assessed in the following functionally distinct muscles: longissimus dorsi (l. dorsi), soleus and rhomboideus. Reductions in plasma concentrations of thyroxine (T4; 32%, P < 0.01), triiodothyronine (T3; 48%, P < 0.001), free T3 (58%, P < 0.001) and hepatic 5'-monodeiodinase (EC 1.11.1.8) activity (74%, P < 0.001) occurred with treatment. Small, although significant, increases in the proportion of type I slow-twitch oxidative fibres occurred with mild hypothyroidism, in l. dorsi (2%, P < 0.01) and soleus (7%, P < 0.01). Nuclear T3-receptor concentration in l. dorsi of hypothyroid animals compared with controls increased by 46% (P < 0.001), a response that may represent a homeostatic mechanism making muscle more sensitive to low levels of circulating thyroid hormones. Nevertheless, Na+, K(+)-ATPase (EC 3.6.1.37) concentration was reduced by 15-16% in all muscles (l. dorsi P < 0.05, soleus P < 0.001, rhomboideus P < 0.05), and Ca(2+)-ATPase (EC 3.6.1.38) concentration was significantly reduced in the two slow-twitch muscles: by 22% in rhomboideus (P < 0.001) and 23% in soleus (P < 0.05). It is concluded that during early postnatal development of large mammals a period of mild hypothyroidism, comparable with that found during undernutrition, induces changes in myofibre differentiation and a down-regulation of cation pumps in skeletal muscle. Such changes would result in slowness of movement and muscle weakness, and also reduce ATP hydrolysis with a concomitant improvement in energetic efficiency.     

Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [125I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [125I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon's horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10(-14)-10(-8) M) and des(1-3) IGF-I (10(-10)-10(-8) M) were found to inhibit whereas IGF-II (10(-14)-10(-8) M) potentiated K+-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.     

Interrater reliability of six tests of trunk muscle function and endurance

Some studies have shown a relationship between trunk muscle strength and low back pain. Measures of trunk muscle strength and endurance, which are feasible in the clinical setting, are needed. The purpose of this study was to determine interrater reliability of six tests of abdominal and trunk extensor muscle strength and endurance. The tests included abdominal and extensor dynamic endurance, hand-held dynamometry of isometric flexion and extension, and abdominal and extensor static endurance. Thirty-nine healthy workers were recruited as subjects. Each was tested by three raters on 3 days within 1 week. Intraclass correlation coefficients (ICC) and the standard error of measurement (SEM) were calculated: abdominal dynamic endurance ICC = .89, SEM = 8 repetitions; extensor dynamic endurance ICC = .78, SEM = 9 repetitions; abdominal isometric force ICC = .25, SEM = 60 N; extensor isometric force ICC = .24, SEM = 68 N; abdominal static endurance ICC = .51, SEM = 35 seconds; extensor static endurance ICC = .59, SEM = 20 seconds. The dynamic endurance tests had acceptable interrater reliability. For the others, reliability was poor and the SEMs were large.