Skeletal Muscle Nuclei Research Articles

Identification and characterization of newt rad (ras associated with diabetes), a gene specifically expressed in regenerating limb muscle.

Formation of the blastema is a key event for limb regeneration in urodele amphibians, and skeletal muscle has been thought to be a major origin of the multipotent blastemal mesenchyme. In the present study, we used differential display to identify the genes expressed differentially in the muscle at the amputation site. We have isolated a cDNA clone that was upregulated during limb regeneration of the Japanese newt, Cynops pyrrhogaster. Deduced amino acid sequence revealed that the cloned cDNA was a newt homolog of rad (ras associated with diabetes), a gene overexpressed in skeletal muscle of Type II diabetic patients. Expression of newt rad (nrad) was not observed in unamputated normal limb muscle, increased within 4 hr after amputation, and then decreased to the level of normal muscle between 11 and 21 days after amputation. In situ hybridization showed that the transcripts of nrad were localized around most of the nuclei of skeletal muscle near the amputation site, indicating the expression of nrad in the multinucleate myotubes. This expression gradually decreased along the distal to proximal axis. No signals were observed in apical epidermal cap or blastemal mesenchyme. However, reverse transcription-PCR analysis detected a very low level of nrad expression in blastema, suggesting the carry-over of nrad expression in blastema from muscle. Administration of retinoic acid, which has been shown to cause an enhanced dedifferentiation in the regenerating limbs, increased nrad expression in more proximally located limb muscle tissues and prolonged the expression period. Thus, it was strongly suggested that the nrad expression is correlated with the dedifferentiation of myotubes of regenerating limbs. We also analyzed the expression of nrad during development. Transcripts were observed in immature oocytes, seen faintly or not seen thereafter until stage 57 when its expression increased again. These results indicated that nrad may play a role(s) in the developmental process as well as limb regeneration.

Emerin

Emerin encoded by the STA gene is the first nuclear protein linked with a muscular dystrophy. Emerin is a 34 kDa, predominantly hydrophilic protein with a single hydrophobic region supposed to serve as a transmembrane domain. It was classified as a type II integral membrane protein localized at the inner nuclear membrane/nuclear lamina with an ubiquitous tissue distribution. It is speculated that emerin is required for the stability and normal function of rigorously moving nuclei in skeletal muscle and heart. During mitosis, emerin is cell-cycle-dependent phosphorylated and shows stage-dependent changes in distribution and localization suggesting that it plays a role in re-assembly of nuclear membranes. Mutations of the emerin gene have been associated with X-linked Emery–Dreifuss muscular dystrophy clinically defined by early joint contractures, progressive muscle weakness, and cardiomyopathy. Hopefully, identification of the protein defect may promote new therapeutic strategies concerning muscle fiber development and stability.

Distribution of emerin and lamins in the heart and implications for Emery-Dreifuss muscular dystrophy.

Emerin is a nuclear membrane protein which is missing or defective in Emery-Dreifuss muscular dystrophy (EDMD). It is one member of a family of lamina-associated proteins which includes LAP1, LAP2 and lamin B receptor (LBR). A panel of 16 monoclonal antibodies (mAbs) has been mapped to six specific sites throughout the emerin molecule using phage-displayed peptide libraries and has been used to localize emerin in human and rabbit heart. Several mAbs against different emerin epitopes did not recognize intercalated discs in the heart, though they recognized cardiomyocyte nuclei strongly, both at the rim and in intranuclear spots or channels. A polyclonal rabbit antiserum against emerin did recognize both nuclear membrane and intercalated discs but, after affinity purification against a pure-emerin band on a western blot, it stained only the nuclear membrane. These results would not be expected if immunostaining at intercalated discs were due to a product of the emerin gene and, therefore, cast some doubt upon the hypothesis that cardiac defects in EDMD are caused by absence of emerin from intercalated discs. Although emerin was abundant in the membranes of cardiomyocyte nuclei, it was absent from many non-myocyte cells in the heart. This distribution of emerin was similar to that of lamin A, a candidate gene for an autosomal form of EDMD. In contrast, lamin B1 was absent from cardiomyocyte nuclei, showing that lamin B1 is not essential for localization of emerin to the nuclear lamina. Lamin B1 is also almost completely absent from skeletal muscle nuclei. In EDMD, the additional absence of lamin B1 from heart and skeletal muscle nuclei which already lack emerin may offer an alternative explanation of why these tissues are particularly affected.

C- myc is expressed in mouse skeletal muscle nuclei during post-natal maturation

Previous data have indicated the presence of c- myc mRNA and protein in mature skeletal muscle, but whether the protein is present as a Myc/Max heterodimer capable of influencing transcription in that tissue or its potential role in pathological muscle tissue has not been examined. The expression of c- myc in normal and mdx dystrophic mouse skeletal muscle was therefore investigated. C- myc mRNA was detected by Northern hybridisation in normal and dystrophic mouse muscle from mice up to 40 days of age and immunohistochemical staining confirmed that c- myc protein was expressed in muscle fibre nuclei in the muscles of mice up to 40 days of age. The presence of Myc-containing complexes that are able to bind to the concensus Myc/Max binding site was demonstrated in these muscles using the electrophoretic mobility shift assay. These results show that c- myc protein is expressed in functional complexes in both normal and dystrophic mouse skeletal muscle during post-natal maturation. They also show that expression of c- myc in mouse skeletal muscle decreases to undetectable levels by about 40 days of age. Although no differences were detected between the expression of c- myc in mdx and control mouse muscle, these data show that muscle contains Myc protein which has previously been demonstrated to be capable of initiating programmed cell death in other tissues.

X‐linked emery‐dreifuss muscular dystrophy can be diagnosed from skin biopsy or blood sample

We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.

H1 histone sub-type distribution and DNA topoisomerase activity in skeletal muscle of tumour-bearing rats

In an investigation of possible causes of the transcriptional defect which has been observed in muscles atrophying in response to tumour growth, the amounts of histone H1 subtypes and the activities of DNA topoisomerase I and II, factors which can affect the structural organisation of chromatin, were studied in nuclei of skeletal muscle of rats bearing a Walker 256 carcinoma. The H1 histones were separated by SDS/PAGE into three fractions, H1.1, H1.2 and H1 o . The level of H1.2 but not that of H1.1 or H1 o was increased in the tumour-bearing animal. Tumour growth did not affect the activity of either DNA topoisomerase I or II.

H1 histone sub-type distribution and DNA topoisomerase activity in skeletal muscle of tumour-bearing rats

In an investigation of possible causes of the transcriptional defect which has been observed in muscles atrophying in response to tumour growth, the amounts of histone H1 subtypes and the activities of DNA topoisomerase I and II, factors which can affect the structural organisation of chromatin, were studied in nuclei of skeletal muscle of rats bearing a Walker 256 carcinoma. The H1 histones were separated by SDS/PAGE into three fractions, H1.1, H1.2 and H1 o. The level of H1.2 but not that of H1.1 or H1 o was increased in the tumour-bearing animal. Tumour growth did not affect the activity of either DNA topoisomerase I or II.

Nerve-Dependent Induction of AChR ϵ-Subunit Gene Expression in Muscle Is Independent of State of Differentiation

To determine if the expression of the ϵ-subunit of the acetylcholine receptor by the subsynaptic nuclei in skeletal muscle is dependent on the state of differentiation of the muscle, we have compared the spatiotemporal distribution of ϵ-subunit transcripts during synapse formation in fetal and adult muscle. Both during "ontogenic" synaptogenesis in the fetus and during "ectopic" synaptogenesis in the adult animal the motor nerve induced locally the expression of the ϵ-subunit mRNA in subsynaptic nuclei. The temporal expression patterns at both types of developing synapses were similar. The results support the view that in muscle developing in vivo ϵ-subunit gene transcription and its stabilization in subsynaptic nuclei is exclusively controlled by the motor neuron, independently of the developmental state of the muscle nuclei. Thus, both nerve and muscle remain plastic in their respective abilities to induce and express the synapse-specific combination of AChR subunit genes.

The Superfamily of Receptors for Growth Hormone, Prolactin, Erythropoietin, and Cytokines

Postnatal developmental pretranslational regulation of skeletal muscle alpha-actin gene expression was investigated. Northern blot analysis of skeletal muscle alpha-actin and beta-tubulin mRNA from 1- and 28-day-old pigs indicated that there are developmental increases in alpha-actin mRNA abundance (P < 0.03) and no significant changes in beta-tubulin mRNA (P > 0.1). A system for isolation of nuclei from porcine skeletal muscle and for transcriptional "run-on" analysis was established in order to investigate the regulatory mechanism of developmental changes in porcine skeletal muscle protein. Skeletal muscle nuclei were isolated from longissimus dorsi (LD) muscle of 1- and 28-day-old pigs by adapting a method to isolate nuclei from cardiac muscle. Results from a [3H]-UTP incorporation assay indicate that these nuclei preparations have the capacity to synthesize RNA and attain maximum incorporation after 40-45 min at 26 degrees C. Messenger RNA syntheses from skeletal muscle nuclei from 1- and 28-day-old pigs were not significantly different (P > 0.25). All nascent tRNA, rRNA, and mRNA in the nuclei were elongated since [3H]-UTP incorporation was reduced after addition of 0.05 micrograms/ml alpha-amanitin to the transcription mixture. Transcription "run-on" assay results indicated that more (P < 0.02) skeletal muscle alpha-actin pre-mRNA was synthesized in the 28-day-old pig skeletal muscle nuclei than in the 1-day-old pig skeletal muscle nuclei. These results indicate that the relative increase in skeletal muscle alpha-actin mRNA observed in the older animals was due, at least in part, to an increase in the transcriptional activity of the skeletal muscle alpha-actin gene.

Detection and characterization of muscle-specific nuclear proteins.

To identify nuclear proteins related to muscle tissue specificity, we tried to prepare antibodies recognizing muscle-specific nuclear proteins. Taking advantage of the autoimmunity of some nuclear proteins, we prepared an antiserum against chick muscle nuclear proteins by injecting protein components of the nuclei isolated from chick breast muscles into breast muscle. Three proteins, named p30, p32, and p37, were detected with the antiserum in a two-dimensional SDS-PAGE pattern of the isolated nuclei. P30 and p32 were not detected in the nuclei of liver, brain, cardiac muscle, or slow type skeletal muscle (anterior latissimus dorsi). They were detected in those of fast type skeletal muscle (pectoralis major and semitendinosus) and smooth muscle (gizzard) at all developmental stages examined. On serial fractionation of muscle cell nuclei, they were detected in a fraction obtained after DNase I treatment of the sample, suggesting that the proteins weakly bind to chromatin. A homology search of amino acid sequences showed that there is no known protein similar to p32.

Trichinella spiralis infected skeletal muscle cells arrest in G2/M and cease muscle gene expression.

Infection by Trichinella spiralis causes a variety of changes in skeletal muscle cells including the hypertrophy of nuclei and decreased expression of muscle specific proteins. Potential cellular processes leading to these changes were investigated. In synchronized muscle infections, [3H]thymidine was incorporated into infected cell nuclei from 2-5 days post infection. Labeled nuclei were stably integrated into the infected cell up to 60 days post infection and appear to originate from differentiated skeletal muscle nuclei present at the time of infection. These nuclei were further shown to contain a mean DNA content of approximately 4N, indicating that the [3H]thymidine uptake reflects DNA synthesis and subsequent long-term suspension of the infected cell in the cell cycle at G2/M. Associated with these changes, muscle specific gene transcripts were reduced to < 1- < 0.1% in the infected cell compared to normal muscle. Transcript levels of the muscle transcriptional regulatory factors myogenin, MyoD1, and Id were reduced to < 10, < 1, and increased approximately 250%, respectively, in the infected cell compared to normal muscle, indicating transcriptional inactivation of muscle genes. DNA synthesis in the infected cell may represent the initiation event which leads to expression of this infected cell phenotype.

Human myocyte-specific enhancer factor 2 comprises a group of tissue-restricted MADS box transcription factors.

The MEF2 site is an essential element of muscle enhancers and promoters that is bound by a nuclear activity found, so far, only in muscle and required for tissue-specific transcription. We have cloned a group of transcription factors from human muscle that are responsible for this activity: They are present in muscle-specific DNA-binding complexes, have a target sequence specificity identical to that of the endogenous activity, and are MEF2 site-dependent transcriptional activators. These MEF2 proteins comprise several alternatively spliced isoforms from one gene and a related factor encoded by a second gene. All share a conserved amino-terminal DNA-binding domain that includes the MADS homology. MEF2 transcripts are ubiquitous but accumulate preferentially in skeletal muscle, heart, and brain. Specific alternatively spliced isoforms are restricted to these tissues, correlating exactly with the presence of endogenous MEF2 activity. Furthermore, MEF2 protein is detected only in skeletal and cardiac muscle nuclei and not in myoblast and nonmuscle cells. Thus, post-transcriptional regulation is important in the generation of tissue-specific MEF2 activity. Cardiac and smooth, as well as skeletal, muscles contain functionally saturating levels of MEF2 trans-activating factors that are absent in nonmuscle cells. Moreover, MEF2 is induced in nonmuscle cells by MyoD; however, MEF2 alone is insufficient to produce the full muscle phenotype. Implications for the molecular mechanisms of myogenesis are considered.

Glucocorticoid-mediated muscle atrophy: alterations in transcriptional activity of skeletal muscle nuclei

Glucocorticoid-induced musle atrophy is associated with a decrease in the level of protein synthesis and a loss of RNA. This paper reports the behaviour of RNA polymerase I- and RNA polymerase II-directed transcription (EC 2.7.7.6) in nuclei isolated from skeletal muscles of rats given a catabolic dose of dexamethasone acetate (5 mg per Kg body weight) over a period of 4 days. Both activities were altered by teh dexamethasone treatment. In the case of RNA polymerase I-mediated transcription there was a loss of template-engaged indicating the existence of an inhbition of initiation of transcription while the rate of elongation of bound enzymes was unaltered. The number of RNA polymerase II-chromatin bound enzymes was increased, but the mean polynucleotide elongation rate was reduced. The possiblity that glucocorticoids may impair the elongation stage of transcription in skeletal muscle by increasing the frequency of premature termination of transcripts is discussed. No evidence was obtained for any increase in ribonuclease activity in muscle nuclei of dexamethasone-treated animals.

Decreased hindlimb skeletal muscle weight and nuclear T 3 receptor binding of obese (ob/ob) mice

Hindlimb muscle weights and binding of L-triiodothyronine (T 3) to isolated nuclei of hindlimb skeletal muscle were investigated in obese (ob/ob) and lean mice. At 12 weeks of age, although weighing substantially more, obese mice had only 55% as much muscle mass as their lean littermates. There was no phenotype effect observed for the equilibrium dissociation constant (Kd). However the apparent binding capacity (Bmax) was significantly less (P<0.001) for obese mice as compared to lean, thus confirming the preliminary results of Khan, et al. (7) which showed a trend for lower T 3 receptor concentration in skeletal muscle nuclei of obese mice.

Interaction of testosterone and testosterone receptor complexes with nuclei of skeletal muscle from intact male mice and from mice bearing the testicular feminization (Tfm) mutant gene.

With the nuclear exchange assay and the nuclear retention assay it is shown that the androgen insensitivity of Tfm mice is probably due to a defect of the nuclear acceptor sites for the testosterone receptor complex. Further on the results obtained point strongly to the possibility that hormone free androgen receptor is localized in the nuclei and in the cytoplasma according to the "equilibrium model". A practicable method for separation of unbound steroids from nuclei is described.

THYROID HORMONE NUCLEAR RECEPTORS IN SKELETAL MUSCLE AS INFLUENCED BY ENVIRONMENTAL TEMPERATURE AND ENERGY INTAKE

Young litter-mate pigs were kept at either 10 or 35 degrees C and fed either a high (H) or a low (L) food intake producing four groups: 10 H, 10 L, 35 H and 35 L. The numbers of receptors for thyroid hormone in the nuclei of skeletal muscle were estimated and found to be greatest in the 35 H group and least in the 10 L group. The numbers of receptors in the 10 H and 35 L groups were similar and took intermediate values. It is suggested that the differences in receptor numbers represent an adaptation which regulates the tissue response to thyroid hormone. Some possible consequences of this adaptation are discussed.

Sodium channels near end‐plates and nuclei of snake skeletal muscle.

1. The spatial distribution of the two predominant types of voltage-gated channels in the sarcolemma, Na+ channels and delayed K+ channels, was studied in skeletal muscle fibres of garter snakes (Thamnophis sirtalis) using loose-seal patch recordings. 2. The average Na+-current density was five times larger in the perijunctional sarcolemma (within 25 microns of the visible edge of the end-plate) than at distant locations. K+ currents were not larger near end-plates. The apparent membrane capacitance, 3-6 microF/cm2, was the same in perijunctional and extrajunctional regions, indicating that differences in Na+-current density reflect differences in the number of Na+ channels per unit membrane area. 3. Perijunctional Na+ channels had the same voltage dependence, gating kinetics and sensitivity to tetrodotoxin as extrajunctional Na+ channels, suggesting that these cells express a single type of Na+ channel. 4. Myonuclei were found to cluster near end-plates and to avoid regions where a nerve branch or blood vessel crossed a fibre's surface. 5. Na+-current density in the sarcolemma above a nucleus was no larger than away from nuclei, indicating that functional Na+ channels are probably not inserted near nuclei. 6. Maps spanning several millimetres of fibre length showed up to sixfold differences in current density between widely separated patches. Differences between patches separated by 50 or 100 microns were much smaller.

Preliminary comparative nuclear T 3 receptor binding characteristics in liver and skeletal muscle of the obese mouse and Zucker fatty rat

L-Triiodothyronine (T 3) binding to isolated nuclei from liver and skeletal muscle of the (ob/ob) mouse and the Zucker rat were examined (obse vs. lean). Present data confirm the earlier report of Guersney and Morishige showing reduced T 3 binding to hepatic nuclei from ob/ob mice. In Zucker rats, however, only 50% of animals indicate the possibility of a difference in hepatic nuclear T 3 receptor concentration between obese and lean. Preliminary results also show a trend for the T 3 receptor concentration to be lower in skeletal muscle nuclei of both (ob/ob) mice and the Zucker rats. Disassociation constant (K d) for the receptor was similar in all tissues examined, suggesting a single population of receptors.

Androgen binding by skeletal-muscle nuclei

A study was made of 3H-19-nortestosterone binding by isolated nuclei and 0.4 M KCl nuclear extract of the rat skeletal muscle. Binding specificity was ascertained by incubation in the presence of various unlabeled steroids. The Kd values were measured for nuclei and 0.4 M KCl nuclear extract (11.6 +/- 2.5 nM and 9.9 +/- 1.6 nM, respectively). The amount of binding sites was 24.1 +/- 1.7 fmol/mg DNA or 13.7 +/- 1.0 fmol/g tissue. Enzymatic treatment with pronase and DNase shows that nuclear androgen receptors are proteins. DNA was noted to have a stabilizing effect. DNase treatment of nuclei during extraction with 0.4 M KCl was shown to significantly increase the amount of specifically bound radioactivity in the extract.

Autoradiographic demonstration of estrogen receptors in the esophagus of baboons

Previous reports have indicated that certain sex steroids are intimately involved in the physiologic activities of the gastrointestinal tract. We performed autoradiographic studies using [3H]estradiol and [3H]dihydrotestosterone on male and female baboons for the purpose of identifying estrogen or androgen receptors, or both, in the lower and midregions of the esophagus. Discrete sites of localization of exposed photographic emulsion were observed over nuclei of skeletal muscle and interstitial cells of male baboons injected with the estrogen. No localization of receptors was observed in these same cells in females given estrogen nor in any males and females injected with the androgen. These observations suggest that skeletal muscle and interstitial cells of the male baboon contain specific highaffinity estrogen receptors that might have a direct effect on the skeletal muscle cells and may modulate their cellular activities.