Developmental expression and location of IGF-I and IGF-II mRNA and protein in skeletal muscle

To investigate the role of IGF in muscle development in vivo, developmental expression and location of IGF-I and -II protein and mRNA were examined in fetal, postnatal, and adult skeletal muscle. Muscle tissue was collected from 30-, 44-, 59-, 68-, 75-, 89-, and 109-d porcine fetuses, 21-d neonatal pigs, and 6-mo-old (adult) pigs. Relative amounts of IGF-II mRNA peaked (P < .05) in 59-d fetal muscle and decreased thereafter. Inversely, muscle IGF-I expression increased (P < .05) to maximal levels around birth. For in situ hybridization, frozen muscle tissue sections (10 microm) were hybridized with a hydrolyzed form of the same riboprobes or incubated with polyclonal or monoclonal antibodies to IGF-I or -II, respectively. The majority of IGF-I and IGF-II mRNA was localized to developing muscle fibers, whereas little signal was found in the surrounding connective tissues. Immunofluorescent localization of IGF-I and -II confirmed that muscle IGF are present in developing muscle fibers. Collectively, these data show that IGF-I and -II are expressed and produced primarily in muscle cells within developing muscle tissue and support the hypothesis that IGF-I and -II modulate fetal muscle development.     

Dihydropyridine receptor gene expression in skeletal muscle from mdx and control mice

The expression of isoform-specific dihydropyrine receptor-calcium channel (DHPR) alpha 1-subunit genes was investigated in mdx and control mouse diaphragm (DIA) and tibialis anterior (TA). RNase protection assays were carried out with a rat DHPR cDNA probe specific for skeletal muscle and a mouse DHPR cDNA probe specific for cardiac muscle. The level of expression of the gene encoding the cardiac DHPR was very weak in TA muscle from both control and mdx mice. Compared to TA, DIA expressed mRNA for the cardiac isoform at significantly higher levels, but mdx and control mouse DIA levels were similar to one another. In contrast, mRNA expression levels for the DHPR skeletal muscle isoform were lower in control DIA than TA. However, there was a dramatic increase in the expression for the DHPR skeletal muscle isoform in mdx DIA compared with control DIA, reaching the TA expression level, whereas dystrophy did not affect TA expression. [3H]-PN200-110 binding was used to further assess DIA DHPR expression at the protein level. The density of binding sites for the probe was not significantly affected in DIA muscles of mdx vs. control mice, but it was reduced in older mdx and control mice. The increase in DHPR mRNA levels without a consequent increase in DHPR protein expression could be secondary to possible enhanced protein degradation which occurs in mdx DIA. The altered DHPR expression levels found here do not appear to be responsible for the severe deficits in contractile function of the mdx DIA.     

Skeletal muscle growth and expression of skeletal muscle alpha-actin mRNA and insulin-like growth factor I mRNA in pigs during feeding and withdrawal of ractopamine

Sixty crossbred barrows were used to study the effect of ractopamine (a phenethanolamine/beta-adrenergic agonist) treatment and its withdrawal on muscle growth and on the relative abundance of skeletal muscle alpha-actin (sk-alpha-actin) mRNA and of liver and longissimus muscle IGF-I mRNA at 4 wk. Ractopamine was fed (20 ppm) for periods of 2, 4, and 6 wk (six pigs per group). Additional pigs (four per group) were fed ractopamine (20 ppm) for 6 wk and then slaughtered 1, 3, and 7 d after withdrawal of ractopamine. Ractopamine increased (P < .05) longisimus muscle weight and protein content, although protein concentrations were not different. The increased muscle weight and protein content attained by feeding ractopamine for 6 wk was retained when ractopamine was withdrawn. The RNA and DNA concentrations did not change, whereas total DNA and RNA content per muscle was 18 and 26.7% greater, respectively, in ractopamine-treated pigs at 4 wk, but there were no differences at 2 or 6 wk or among the withdrawal groups. The relative abundance of sk-alpha-actin mRNA in the longissimus muscle was 41 and 62% greater (P < .05) in treated animals at 2 and 4 wk but was similar to that in controls at 6 wk and during the withdrawal period. The relative abundance of IGF-I mRNA in liver and longissimus muscle was not altered with ractopamine treatment for 4 wk. These results indicate that the ractopamine-enhanced muscle growth may result from increased myofibrillar gene expression at the pretranslational level, which is maximal with short-term treatment of ractopamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle

Many properties of skeletal muscle cells are closely regulated by motor nerves. Neuromuscular synaptic transmission (including the 'activity' it triggers) mediates many of these effects, while denervation results in a different spectrum of muscle cell changes. However, little is known about the early regulatory events that occur in mature muscle cells in response to muscle activity or denervation. We have examined the effects of motor nerve stimulation and denervation on the expression of 4 immediate early genes (IEGs)--c-jun, junB, zif268, and nur77--in mature mouse gastrocnemius muscle. Electrical stimulation of the sciatic nerve in a pattern of brisk intermittent exercise induced a marked rise in zif268 and c-jun mRNA levels within 45 min, a minimal rise in junB, and no change in nur77 mRNA levels. By contrast, surgical denervation resulted in a marked increase of c-jun, a slight rise in junB, and no change in nur77 or zif268 mRNA levels. These findings show that neural stimulation and denervation lead to differential patterns of IEG expression. The selectivity of these patterns suggests that differential IEG expression may play an important role in regulating the specific phenotypic changes in skeletal muscles that result from denervation, innervation, and various patterns of stimulation.     

Abnormal expression of laminin beta 1 chain in skeletal muscle of adult-onset limb-girdle muscular dystrophy

BACKGROUND: Laminin 2 is a major component of the basal lamina of skeletal muscle cells. It is a heterotrimer composed of 3 chains: merosin (laminin alpha 2 chain), beta 1, and gamma 1. Deficiency of merosin, with or without laminin beta 1 chain reduction, is associated with some forms of congenital muscular dystrophy. Deficient expression of laminin beta 1 chain is also associated with some cases of merosin-positive congenital muscular dystrophy. The expression of laminin 2 subunits has not been well studied in the skeletal muscle of limb-girdle muscular dystrophy (LGMD), nor has much attention been given to the significance of reduction of individual laminin 2 subunits, such as beta 1. OBJECTIVES: To examine the expression of laminin 2 subunits in skeletal muscle in patients with LGMD and to define the clinical features of patients with LGMD who have abnormal expression of laminin 2 subunits. METHODS: We studied muscle biopsy specimens from 18 patients with LGMD using immunofluorescence with antibodies against dystrophin C-terminus, beta-dystroglycan, alpha-sarcoglycan, gamma-sarcoglycan, and the laminin subunits merosin, beta 1, and gamma 1. Of the 18 biopsy specimens, 9 were available for electron microscopic examination of the muscle basement membrane. The clinical features associated with abnormal laminin beta 1 chain immunoreactivity were further described. RESULTS: Laminin beta 1 chain was either barely detectable or severely reduced in 3 cases of patients with LGMD in which the biopsy specimens showed normal staining with the other antibodies. Patients in all 3 cases had common clinical features consistent with a slowly progressive, adult-onset LGMD. Specimens from 2 of the 3 cases that were available for ultrastructural examination showed significant abnormalities of the muscle fiber basement membrane. CONCLUSIONS: Abnormal expression of laminin beta 1 chain without concomitant deficiency of alpha-sarcoglycan in skeletal muscle has not been previously described in LGMD. Reduced laminin beta 1 chain immunoreactivity may potentially serve as a marker for defining subsets of individuals with LGMD, in particular those with slowly progressive, adult-onset pelvifemoral presentation. The abnormality of muscle fiber basement membranes in specimens from cases that were available for ultrastructural study suggests that defects in the extracellular matrix may play a role in the pathogenesis of this subset of LGMD.     

Chronic loss of glucocorticoids following adrenalectomy down-regulates the expression of glucocorticoid receptor mRNA in the rat forebrain

In the negative feedback model, loss of endogenous glucocorticoids up-regulates the expression of glucocorticoid receptor mRNA. To elucidate further the effect of chronic lack of glucocorticoids on the expression of glucocorticoid receptor mRNA and protein, in situ hybridization and immunohistochemical methods were used to examine the long-term alteration of glucocorticoid receptor mRNA and its immunoreactivity in the forebrain of adrenalectomized rats. Constant lack of glucocorticoids resulted in marked decrease in the expression of glucocorticoid receptor mRNA and disappearance of glucocorticoid receptor immunoreactivity in many forebrain structures. In particular, in the suprapyramidal blade of the hippocampal granule cell layer and cerebral cortex, many cells showed almost no glucocorticoid receptor mRNA signals. These results suggest that long-term loss of endogenous glucocorticoids down-regulates the levels of glucocorticoid receptor mRNA, leading to reduction in the synthesis of glucocorticoid receptors in the rat forebrain. Therefore, the presence of endogenous glucocorticoids is vital to the continued expression of glucocorticoid receptor mRNA.     

Molecular cloning and functional expression of a skeletal muscle dihydropyridine receptor from Rana catesbeiana

In skeletal muscle the dihydropyridine receptor is the voltage sensor for excitation-contraction coupling and an L-type Ca2+ channel. We cloned a dihydropyridine receptor (named Fgalpha1S) from frog skeletal muscle, where excitation-contraction coupling has been studied most extensively. Fgalpha1S contains 5600 base pairs coding for 1688 amino acids. It is highly homologous with, and of the same length as, the C-truncated form predominant in rabbit muscle. The primary sequence has every feature needed to be an L-type Ca2+ channel and a skeletal-type voltage sensor. Currents expressed in tsA201 cells had rapid activation (5-10 ms half-time) and Ca2+-dependent inactivation. Although functional expression of the full Fgalpha1S was difficult, the chimera consisting of Fgalpha1S domain I in the rabbit cardiac Ca channel had high expression and a rapidly activating current. The slow native activation is therefore not determined solely by the alpha1 subunit sequence. Its Ca2+-dependent inactivation strengthens the notion that in rabbit skeletal muscle this capability is inhibited by a C-terminal stretch (Adams, B., and Tanabe, T. (1997) J. Gen. Physiol. 110, 379-389). This molecule constitutes a new tool for studies of excitation-contraction coupling, gating, modulation, and gene expression.     

Differential titin isoform expression in human skeletal muscle

Mammalian skeletal muscle expresses at least two isoforms of the cytoskeletal protein titin (connectin; MW approximately 3000 kDa). These isoforms are associated with different passive force curves, and thus may affect physical performance. To study the distribution of titin and its possible influence on performance in humans, muscle biopsies were obtained from 15 males (mean +/- SE; age = 25.4 +/- 2.9 years, height = 177.7 +/- 1.8 cm, weight = 76.5 +/- 2.2 kg). Two biopsies were obtained on separate occasions from both the right and left vastus lateralis, and one biopsy each from the lateral head of the right gastrocnemius and the right soleus, with all biopsies handled identically. Fibre type analyses were performed via mATPase histochemistry. Expression of titin and myosin heavy chain isoforms were determined by SDS-PAGE. Titin bands in the resulting gels were highly repeatable and were verified by migration patterns, as well as Western blot analysis. Two groups of subjects were identified: group 1 (n = 10) expressed only one titin isoform (titin-1) in all biopsies, and group 2 (n = 5) expressed two titin isoforms (titin-1 and titin-2) in all biopsies. No significant differences (P > 0.05) between groups were observed for percentage fibre types, percentage fibre type areas, fibre type cross-sectional areas, and percentage myosin heavy chain expression when comparing individual muscles, sampling times or bilateral comparisons. This is the first report of differential titin isoform expression in healthy, mature human skeletal muscle, but it is not clear why this occurs or what influence this may have on performance.     

Alpha 1 adrenergic receptor activation of proto-oncogene expression in arterial smooth muscle: regulation by nitric oxide and vascular injury

The role of the endothelium in modulating smooth muscle cell growth is unclear. alpha 1 adrenergic receptors activate proto-oncogene expression in smooth muscle. We have now found in rat aorta that carbachol, a muscarinic cholinergic agonist that promotes release of nitric oxide (NO), inhibits expression of c-fos and c-jun mRNA induced by alpha 1 receptors. NO synthase inhibitors blocked the effects of carbachol on c-fos mRNA and a cGMP analog mimicked carbachol. After balloon injury in rat aorta using in situ hybridization, the catecholamine-induced increase in c-fos mRNA expression in the medial layer was inhibited by the alpha 1 receptor antagonists, prazosin and chloroethylclonidine. In the neointima, this response was fully blocked by prazosin; however, chloroethylclonidine only partially inhibited it. These results suggest that NO, acting through a cGMP-dependent mechanism, inhibits expression of the c-fos and c-jun genes in arteries, which may contribute to the growth-inhibiting effects of the endothelium. After endothelial damage, the activation of c-fos in neointima by adrenergic stimulation may involve a subtype of alpha 1 receptor different from that utilized in medial smooth muscle.     

Coronary artery and cultured aortic smooth muscle cells express mRNA for both the classical estrogen receptor and the newly described estrogen receptor beta

Estrogens exhibit potent anti-atherogenic effects through mechanisms which may involve direct effects on the artery. The existence of the classical estrogen receptor (ERalpha) in vascular tissues has been established. Recently a new estrogen receptor (ERbeta) has been discovered which represents a distinct gene product with homology to the classical ERalpha. The purpose of the present study was to determine if ERbeta mRNA is expressed in vascular tissues of female and male primates. Oligonucleotide primers were developed for the specific RT-PCR amplification of ERalpha or ERbeta mRNA. RT-PCR products of the appropriate size for ERalpha and for ERbeta were observed after amplification of RNA isolated from coronary arteries of both male and female cynomolgus monkeys. Similar results were obtained from cultured aortic smooth muscle cells and from monkey reproductive tissues such as ovary and uterus. The relative expression of ERbeta to ERalpha mRNA was greatest in ovary, on the same order of magnitude in monkey vascular tissues and uterus, while the human breast cancer cell line MCF-7 exhibited a very low level of ERbeta relative to ERalpha. Sequence analysis of isolated RT-PCR products showed >95% similarity between the monkey and the published human sequences for both ERalpha and ERbeta. These findings suggest that estrogen may influence vascular gene expression not only through classical ERalpha but also through the newly described ERbeta. These findings also demonstrate the potential for targeting of these receptors in males for prevention or treatment of heart disease.     

Desmin-lacZ transgene expression and regeneration within skeletal muscle transplants

The purpose of this study was to investigate the initiation and time course of the regeneration process in fragments of skeletal muscle transplants as a function of muscle tissue age at implantation. The appearance of desmin occurs at the very beginning of myogenesis. The transgenic desmin nls lacZ mice used in the study bear a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coli beta-galactosidase. The desmin lacZ transgene labels muscle cells in which the desmin synthesis programme has commenced. We implanted pectoralis muscle fragments from fetal transgenic embryos and mature and old transgenic mice into mature non-transgenic mice. Early events of myogenesis occurring during regeneration started sooner in transplants from 4-month-old (day 3 post-implantation) muscle than in those from 24-month-old (day 5-6 post-implantation) muscle, and they lasted longer in those from young (day 17 post-implantation) than in those from old (day 14 post-implantation) muscle fragments. In adult muscle, transgene activation proceeded from the periphery toward the centre of the transplant. In transplants from fetal 18-day-old pectoralis, myotubes with transgene activity were observed from day 1 to day 19. Desmin immunoreactivity, which appeared about one day after transgene activation, was followed by myosin expression. In adult transplants, the continuity of laminin labelling was disrupted around degenerative fibres, illustrating alteration of the extracellular matrix. Our data suggest that satellite cells from old muscle tissue have lower proliferative capacity and/or less access to trophic substances released by the host (damaged fibres, vascularization) than those from fetal or young adult muscle.     

Modulation of glucocorticoid receptor gene expression by antidepressant drugs

The literature on the setting mechanisms of dental amalgams made from powders of silver-rich alloys of tin and/or copper has been critically reviewed. In Part 1 of the review, the microstructure and phase content of recently set amalgams are described. The composition, morphology, and location of product phases are emphasized, since these features are clues to the setting reaction. Thus, Part 1 provides the background needed to understand the kinetics of the setting reactions, which is the topic of Part 2 of the review.     

Human cytomegalovirus increases modified low density lipoprotein uptake and scavenger receptor mRNA expression in vascular smooth muscle cells

Evidence suggests a possible role for human cytomegalovirus (HCMV) in the development of arteriosclerosis. One of the earliest events in plaque formation is the accumulation of lipid-laden foam cells, derived from macrophages and smooth muscle cells (SMCs). The lipid accumulation that occurs depends upon the uptake of oxidized LDL (Ox-LDL), a process in which the scavenger receptor (SR) has been postulated to play an important role. We therefore examined the effects of HCMV on this process. We demonstrate that HCMV infection of human SMCs increases modified LDL uptake and stimulates class A SR gene (SR-A) mRNA expression. In addition, infection of rat SMCs with HCMV, which causes immediate early gene expression (IE72/IE84), but no early or late HCMV gene products and no cytopathic effects, also increases SMC uptake of Ox-LDL and acetylated LDL, with either effect blocked by an excess of either cold Ox-LDL or acetylated-LDL, and by fucoidin, an SR competitor. Cotransfection of an IE72, but not an IE84, expression plasmid and a plasmid containing a Class A SR promoter/reporter gene construct enhances SR promoter activity. Since increased Ox-LDL uptake is believed to play an important role in arteriosclerosis, these results provide a link between HCMV infection and arteriosclerotic plaque formation.     

Regulation of glucocorticoid receptor mRNA in nasal mucosa by local administration of fluticasone and budesonide

The glucocorticoid receptor (GR) is downregulated by glucocorticoids (autoregulation). In contrast, the metallothionein gene (MTIIa) is positively regulated by glucocorticoids, which requires a functional receptor protein. We have investigated the expression of GR and MTIIa mRNA in nasal mucosal biopsy specimens, nasal brush-lavage samples, and peripheral blood lymphocytes from 14 healthy volunteers after local treatment with one of two different glucocorticoids: fluticasone propionate or budesonide. In nasal mucosal biopsy specimens, a significant decrease in GR mRNA occurred with increasing doses of both steroids, whereas a significant and parallel increase in MTIIa mRNA was observed. We found nasal brush-lavage less suitable for studies of GR mRNA and MTIIa mRNA regulation by locally administered glucocorticoids. In mucosal biopsy specimens, but not in peripheral blood lymphocytes, we found a correlation between basal GR mRNA and MTIIa mRNA levels, where low GR mRNA levels were associated with low MTIIa mRNA levels, and vice versa. In conclusion, this study shows that locally administered glucocorticoids significantly affect the expression of specific genes and that there is an interindividual and tissue-specific variation in GR mRNA and MTIIa mRNA expression, which may be used in studies of variations in clinical responses to nasal glucocorticoids.     

Early adversity and serotonin transporter genotype interact with hippocampal glucocorticoid receptor mRNA expression, corticosterone, and behavior in adult male rats.

Despite its importance for development, relatively little is known about how allelic variation interacts with both pre- and postnatal stress. We examined the interaction between serotonin transporter (5-HTT) genotype, prenatal and postnatal stress on glucocorticoid receptor (GR) mRNA expression, corticosteroid stress responses, and behavior in adult male rats. Prenatal stress involved a daily restraint of pregnant dams from gestational Day 10–21. Postnatal stress involved raising pups after parturition either by their mothers (MR) or in the artificial rearing (AR) paradigm, with or without additional “licking-like” stroking stimulation. 5-HTT genotype, hippocampal GR mRNA level, corticosteroid stress response, and behaviors including startle response, prepulse inhibition (PPI), and locomotor activity were measured in adult male rat offspring. We found significant genotype by prenatal stress interactions for hippocampal GR mRNA levels and for the corticosterone stress responses in adulthood. In contrast, behavioral endpoints tended to be more clearly affected by an interaction between genotype and postnatal environment. These findings suggest that allelic variation in the 5-HTT gene interacts with the prenatal environment to affect the hypothalamic-pituitary-adrenal (HPA) axis physiology and the postnatal environment to affect behavior. These results are the first to indicate a role for genetic variation in the 5-HTT gene in physiology and behavior in the rat. (PsycINFO Database Record (c) 2011 APA, all rights reserved)