Expression Of Muscle Regulatory Factors Research Articles

LncRNA GPRC5D-AS1 as a ceRNA inhibits skeletal muscle aging by regulating miR-520d-5p.

Sarcopenia induced by muscle aging is associated with negative outcomes in a variety of diseases. Long non-coding RNAs are a class of RNAs longer than 200 nucleotides with lower protein coding potential. An increasing number of studies have shown that lncRNAs play a vital role in skeletal muscle development. According to our previous research, lncRNA GPRC5D-AS1 is selected in the present study as the target gene to further study its effect on skeletal muscle aging in a dexamethasone-induced human muscle atrophy cell model. As a result, GPRC5D-AS1 functions as a ceRNA of miR-520d-5p to repress cell apoptosis and regulate the expression of muscle regulatory factors, including MyoD, MyoG, Mef2c and Myf5, thus accelerating myoblast proliferation and differentiation, facilitating development of skeletal muscle. In conclusion, lncRNA GPRC5D-AS1 could be a novel therapeutic target for treating sarcopenia.

A clinical herbal prescription Gu-Shu-Kang capsule exerted beneficial effects on the musculoskeletal system of dexamethasone-treated mice by acting on tissue IGF-1 signalling pathway

Context Gu-Shu-Kang (GSK) is a clinical traditional Chinese medicine prescription for the treatment of primary osteoporosis. Objective This study investigates the protection of GSK against dexamethasone (Dex)-induced disturbance of musculoskeletal system in male mice and to identify the underlying mechanism. Materials and methods Male C57BL/6 mice in Dex-treated groups were orally administered (i.g.) with vehicle, low dose (0.38 g/kg), middle dose (0.76 g/kg), or high dose (1.52 g/kg) of GSK for 8 weeks. A control group was designed without any treatment. The quadriceps femoris, tibialis anterior and gastrocnemius were harvested. Molecular expression was determined by RT-PCR and immunoblotting. Results Treatment with GSK enhanced weight-loaded swimming time (from 411.7 ± 58.4 s in Dex group to 771.4 ± 87.3 s in GSK-M) and grip strength (from 357.8 ± 23.9 g in Dex group to 880.3 ± 47.6 g in GSK-M). GSK produced a rise in cross-sectional area of myofibers and promoted a switching of glycolytic-to-oxidative myofiber. The administration with GSK affected expression of muscle regulatory factors shown by the down-regulation in MuRF-1 and atrogin-1 and the up-regulation in myogenic differentiation factor (MyoD) and myosin heavy chain (MHC). GSK stimulated tissue IGF-1 signalling pathway (IGF-1R/PI3K/Akt), not only in skeletal muscle but also in bone associated with the amelioration of trabecular bone mineral density and the improvement of osteogenesis. Conclusions These findings revealed the potential mechanisms involved in the beneficial effects of Gu-Shu-Kang on musculoskeletal system in mice with challenging to dexamethasone, and this prescription may have applications in management for muscle atrophy and osteoporosis triggered by glucocorticoid.

Effects of feeding level and sexual maturation on expression of genes regulating growth mechanisms in rainbow trout (Oncorhynchus mykiss)

The energy and nutrient demands of sexual maturation in salmonids requires a shift from somatic growth to gonadal development, although a loss of skeletal muscle can be avoided with adequate nutrition. How nutrient availability or ration affects the endocrine and growth factor systems that integrate nutrient partitioning, muscle metabolism, and growth during sexual maturation is poorly understood. Primary objectives of the current study were to determine how sexual maturation and ration affect expression of estrogen receptors, components of the GH/IGF axis and TGFβ superfamily system, and muscle specific regulatory factors during gonadal development in female rainbow trout (Oncorhynchus mykiss). Female diploid rainbow trout approaching ovulation were fed for 12 weeks at 0.25% and 0.50% tank biomass, and to apparent satiation. Controls for the effects of gonadal development included sterile triploid trout and two diploid females that failed to undergo gonadal development. Expression of era1 was increased in muscle of maturing diploids suggesting an increase in the catabolic effects of 17β-estradiol (E2) on muscle. In the liver, expression of era2 increased with maturation, but expression of all estrogen receptors were increased with higher rations suggesting improved E2 sensitivity for vitellogenin production. Hepatic expression of ghrs and igfs were generally reduced with maturation suggesting a reduction in systemic growth promotion. Increased ration improved capacity for IGF signaling in muscle via increased igf1 and igfr1b expression, potentially lessening effects of reduced circulating IGF-I on muscle growth. The primary carrier of IGFs, hepatic igfbp2b1, increased with maturation likely reducing systemic IGF activity, and was also elevated with higher rations. All other hepatic IGF binding proteins, except igfbp1, showed some reduction with maturation and several increased with increased ration. Increased muscle igfbp6b1 suggests restricted myogenesis with maturation. Reductions in expression of muscle regulatory factors with maturation including pax7a, myog and myf5, support reduced myogenesis with maturation but were unaffected by ration. In addition, although mstn2a was the only myostatin gene affected by maturation or ration, altered expression of components of the TGFβ superfamily suggest increased ration may inhibit myostatin signaling. In summary, moderate feeding appears to counter many changes in endocrine and growth factor system gene expression associated with growth reduction during maturation, perhaps altering endocrine and growth factor regulation to spare muscle atrophy and better prepare the fish for post-spawning recovery.

Anemic hypoxemia reduces myoblast proliferation and muscle growth in late-gestation fetal sheep.

Fetal skeletal muscle growth requires myoblast proliferation, differentiation, and fusion into myofibers in addition to protein accretion for fiber hypertrophy. Oxygen is an important regulator of this process. Therefore, we hypothesized that fetal anemic hypoxemia would inhibit skeletal muscle growth. Studies were performed in late-gestation fetal sheep that were bled to anemic and therefore hypoxemic conditions beginning at ∼125 days of gestation (term = 148 days) for 9 ± 0 days (n = 19) and compared with control fetuses (n = 16). A metabolic study was performed on gestational day ∼134 to measure fetal protein kinetic rates. Myoblast proliferation and myofiber area were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. mRNA expression of muscle regulatory factors was determined in BF. Fetal arterial hematocrit and oxygen content were 28% and 52% lower, respectively, in anemic fetuses. Fetal weight and whole body protein synthesis, breakdown, and accretion rates were not different between groups. Hindlimb length, however, was 7% shorter in anemic fetuses. TA and FDS muscles weighed less, and FDS myofiber area was smaller in anemic fetuses compared with controls. The percentage of Pax7+ myoblasts that expressed Ki67 was lower in BF and tended to be lower in FDS from anemic fetuses indicating reduced myoblast proliferation. There was less MYOD and MYF6 mRNA expression in anemic versus control BF consistent with reduced myoblast differentiation. These results indicate that fetal anemic hypoxemia reduced muscle growth. We speculate that fetal muscle growth may be improved by strategies that increase oxygen availability.

Preserved Capacity for Adaptations in Strength and Muscle Regulatory Factors in Elderly in Response to Resistance Exercise Training and Deconditioning.

Aging is related to an inevitable loss of muscle mass and strength. The mechanisms behind age-related loss of muscle tissue are not fully understood but may, among other things, be induced by age-related differences in myogenic regulatory factors. Resistance exercise training and deconditioning offers a model to investigate differences in myogenic regulatory factors that may be important for age-related loss of muscle mass and strength. Nine elderly (82 ± 7 years old) and nine young, healthy persons (22 ± 2 years old) participated in the study. Exercise consisted of six weeks of resistance training of the quadriceps muscle followed by eight weeks of deconditioning. Muscle biopsy samples before and after training and during the deconditioning period were analyzed for MyoD, myogenin, insulin-like growth-factor I receptor, activin receptor IIB, smad2, porin, and citrate synthase. Muscle strength improved with resistance training by 78% (95.0 ± 22.0 kg) in the elderly to a similar extent as in the young participants (83.5%; 178.2 ± 44.2 kg) and returned to baseline in both groups after eight weeks of deconditioning. No difference was seen in expression of muscle regulatory factors between elderly and young in response to exercise training and deconditioning. In conclusion, the capacity to gain muscle strength with resistance exercise training in elderly was not impaired, highlighting this as a potent tool to combat age-related loss of muscle function, possibly due to preserved regulation of myogenic factors in elderly compared with young muscle.

Feeding gelatinized carbohydrate in the diets of magur,Clarias batrachus(Linnaeus, 1758): Effects on growth performance, enzyme activities and expression of muscle regulatory factors

Feeding gelatinized carbohydrate in the diets of magur,<i>Clarias batrachus</i>(Linnaeus, 1758): Effects on growth performance, enzyme activities and expression of muscle regulatory factors

CCAAT/Enhancer Binding Protein \u03b2 inhibits myogenic differentiation via ID3

Myogenesis is regulated by the coordinated expression of muscle regulatory factors, a family of transcription factors that includes MYOD, MYF5, myogenin and MRF4. Muscle regulatory factors are basic helix-loop-helix transcription factors that heterodimerize with E proteins to bind the regulatory regions of target genes. Their activity can be inhibited by members of the Inhibitor of DNA binding and differentiation (ID) family, which bind E-proteins with high affinity, thereby preventing muscle regulatory factor-dependent transcriptional responses. CCAAT/Enhancer Binding protein beta (C/EBPβ) is a transcription factor expressed in myogenic precursor cells that acts to inhibit myogenic differentiation, though the mechanism remains poorly understood. We identify Id3 as a novel C/EBPβ target gene that inhibits myogenic differentiation. Overexpression of C/EBPβ stimulates Id3 mRNA and protein expression, and is required for C/EBPβ-mediated inhibition of myogenic differentiation. Misexpression of C/EBPβ in myogenic precursors, such as in models of cancer cachexia, prevents the differentiation of myogenic precursors and we show that loss of Id3 rescues differentiation under these conditions, suggesting that the stimulation of Id3 expression by C/EBPβ is an important mechanism by which C/EBPβ inhibits myogenic differentiation.

Lamina-associated polypeptide 1 is dispensable for embryonic myogenesis but required for postnatal skeletal muscle growth.

Lamina-associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that has been implicated in striated muscle maintenance. Mutations in its gene have been linked to muscular dystrophy and cardiomyopathy. As germline deletion of the gene encoding LAP1 is perinatal lethal, we explored its potential role in myogenic differentiation and development by generating a conditional knockout mouse in which the protein is depleted from muscle progenitors at embryonic day 8.5 (Myf5-Lap1CKO mice). Although cultured myoblasts lacking LAP1 demonstrated defective terminal differentiation and altered expression of muscle regulatory factors, embryonic myogenesis and formation of skeletal muscle occurred in both mice with a Lap1 germline deletion and Myf5-Lap1CKO mice. However, skeletal muscle fibres were hypotrophic and their nuclei were morphologically abnormal with a wider perinuclear space than normal myonuclei. Myf5-Lap1CKO mouse skeletal muscle contained fewer satellite cells than normal and these cells had evidence of reduced myogenic potential. Abnormalities in signalling pathways required for postnatal hypertrophic growth were also observed in skeletal muscles of these mice. Our results demonstrate that early embryonic depletion of LAP1 does not impair myogenesis but that it is necessary for postnatal skeletal muscle growth.

Age-related changes affect rat rotator cuff muscle function

The influence of age on rotator cuff function and muscle structure remains poorly understood. We hypothesize that normal aging influences rotator cuff function, muscle structure, and regulatory protein expression in an established rat model of aging. Seventeen rats were obtained from the National Institute on Aging. The supraspinatus muscles in 11 middle-aged (12 months old) and 6 old (28 months old) rats were studied for age-related changes in rotator cuff neuromuscular function by in vivo muscle force testing and electromyography (EMG). Changes in muscle structure and molecular changes were assessed with quantitative immunohistochemistry for myogenic determination factor 1 (MyoD) and myogenic factor 5 (Myf5) expression. Old animals revealed significantly decreased peak tetanic muscle force at 0.5 N and 0.7 N preload tension (P < .05). The age of the animal accounted for 20.9% of variance and significantly influenced muscle force (P = .026). Preload tension significantly influenced muscle force production (P < .001) and accounted for 12.7% of total variance. There was regional heterogeneity in maximal compound motor action potential (CMAP) amplitude in the supraspinatus muscle; the proximal portion had a significantly higher CMAP than the middle and distal portions (P < .05). The expression of muscle regulatory factors MyoD and Myf5 was significantly decreased in old animals compared with middle-aged animals (P < .05). The normal aging process in this rat model significantly influenced contractile strength of the supraspinatus muscle and led to decreased expression of muscle regulatory factors. High preload tensions led to a significant decrease in force production in both middle-aged and old animals.

TFE3 inhibits myoblast differentiation in C2C12 cells via down-regulating gene expression of myogenin

Transcription factor E3 (TFE3) belongs to a basic helix–loop–helix family, and is involved in the biology of osteoclasts, melanocytes and their malignancies. We previously reported the metabolic effects of TFE3 on insulin in the liver and skeletal muscles in animal models. In the present study, we explored a novel role for TFE3 in a skeletal muscle cell line. When TFE3 was overexpressed in C2C12 myoblasts by adenovirus before induction of differentiation, myogenic differentiation of C2C12 cells was significantly inhibited. Adenovirus-mediated TFE3 overexpression also suppressed the gene expression of muscle regulatory factors (MRFs), such as MyoD and myogenin, during C2C12 differentiation. In contrast, knockdown of TFE3 using adenovirus encoding short-hairpin RNAi specific for TFE3 dramatically promoted myoblast differentiation associated with significantly increased expression of MRFs. Consistent with these findings, promoter analyses via luciferase reporter assay and electrophoretic mobility shift assay suggested that TFE3 negatively regulated myogenin promoter activity by direct binding to an E-box, E2, in the myogenin promoter. These findings indicated that TFE3 has a regulatory role in myoblast differentiation, and that transcriptional suppression of myogenin expression may be part of the mechanism of action.

C2c12 Skeletal Muscle Satellite Cells Are Divergently Responsive To Different Physiologically Relevant Doses Of Interleukin-6

Interleukin-6 (IL-6) is elevated to varying extents in skeletal muscle and the circulation in response to stressors, including endurance exercise and muscle injury. We hypothesized that IL-6 may influence proteins involved in mitochondrial function or satellite cell function, or both. Further, these effects may be downstream of IL-6 binding to gp130, inducing JAK activation and STAT3 phosphorylation. PURPOSE: To probe if the effects of IL-6 on selected parameters of metabolic and regenerative function, are dependent on dose and are related to the gp130 signaling cascade. METHODS: C2C12 myoblasts (immortalised murine skeletal muscle satellite cells) were cultured under standard conditions promoting proliferation (10% fetal calf serum) in the presence or absence of low, moderate or high doses of IL-6 (10 pg/ml; 100 pg/ml; 10 ng/ml) administered at 0 and 24 hours. Cells were harvested at 48 hours and analysed for cell viability (trypan blue), mitochondrial viability and function (MTT), proliferation (cell count), muscle regulatory factor expression (MyoD) and selected intracellular signaling proteins (total and phospho-STAT3). RESULTS: Cell viability and proliferation were not affected at any dose. Mitochondrial dehydrogenase function (MTT) was elevated only by low dose (1.14 fold increase, P<0.05). In contrast, MyoD, an early pro-differentiation muscle regulatory factor was significantly elevated in response to high dose IL-6 (1.4 fold increase P<0.05, with a tendency to respond to medium dose, 1.3 fold). Total STAT3 protein was elevated, only with low dose IL-6 (1.9 fold increase, P<0.01), but phospho-STAT3 declined as IL-6 dose increased (decreased to 0.80, 0.60 and further to 0.33 of control, P<0.01 to P<0.005). CONCLUSIONS: These data lead to the conclusion that the C2C12 skeletal muscle satellite cell line is responsive to IL-6 concentrations across the physiological range, but responses differ for low vs. high doses. Exposure to IL-6 for 48 hours was sufficient to alter cell signaling downstream of the gp130 receptor, but also with a divergent dose-dependence. Therefore, it is essential to consider the physiologically relevant circulating and tissue cytokine concentrations in vivo, when probing the cellular response to IL-6 in vitro. Supported by the South African Medical Research Council.

A distinct profile of myogenic regulatory factor detection within Pax7+ cells at S phase supports a unique role of Myf5 during posthatch chicken myogenesis

Satellite cells are skeletal muscle stem cells that provide myogenic progeny for myofiber growth and repair. Temporal expression of muscle regulatory factors (MRFs) and the paired box transcription factor Pax7 defines characteristic phases of proliferation (Pax7(+)/MyoD(+)/myogenin(-)) and differentiation (Pax7(-)/MyoD(+)/myogenin(+)) during myogenesis of satellite cells. Here, using bromodeoxyuridine (BrdU) labeling and triple immunodetection, we analyzed expression patterns of Pax7 and the MRFs MyoD, Myf5, or myogenin within S phase myoblasts prepared from posthatch chicken muscle. Essentially, all BrdU incorporation was restricted to Pax7(+) cells, of which the majority also expressed MyoD. The presence of a minor BrdU(+)/Pax7(+)/myogenin(+) population in proliferation stage cultures suggests that myogenin up-regulation is alone insufficient for terminal differentiation. Myf5 was detected strictly within Pax7(+) cells and decreased during S phase while MyoD presence persisted in cycling cells. This study provides novel data in support of a unique role for Myf5 during posthatch myogenesis.

Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133

The expression of three microRNAs, miR-1, miR-206 and miR-133 is restricted to skeletal myoblasts and cardiac tissue during embryo development and muscle cell differentiation, which suggests a regulation by muscle regulatory factors (MRFs). Here we show that inhibition of C2C12 muscle cell differentiation by FGFs, which interferes with the activity of MRFs, suppressed the expression of miR-1, miR-206 and miR-133. To further investigate the role of myogenic regulators (MRFs), Myf5, MyoD, Myogenin and MRF4 in the regulation of muscle specific microRNAs we performed gain and loss-of-function experiments in vivo, in chicken and mouse embryos. We found that directed expression of MRFs in the neural tube of chicken embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in a loss of miR-1 and miR-206 expression. Taken together our results demonstrate differential requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis.

Myogenic differentiation during regrowth of atrophied skeletal muscle is associated with inactivation of GSK-3β

Muscle atrophy contributes to morbidity and mortality in aging and chronic disease, emphasizing the need to gain understanding of the mechanisms involved in muscle atrophy and (re)growth. We hypothesized that the magnitude of muscle regrowth during recovery from atrophy determines whether myonuclear accretion and myogenic differentiation are required and that insulin-like growth factor (IGF)-I/Akt/glycogen synthase kinase (GSK)-3beta signaling differs between regrowth responses. To address this hypothesis we subjected mice to hindlimb suspension (HS) to induce atrophy of soleus (-40%) and plantaris (-27%) muscle. Reloading-induced muscle regrowth was complete after 14 days and involved an increase in IGF-IEa mRNA expression that coincided with Akt phosphorylation in both muscles. In contrast, phosphorylation and inactivation of GSK-3beta were observed during soleus regrowth only. Furthermore, soleus but not plantaris regrowth involved muscle regeneration based on a transient increase in expression of histone 3.2 and myosin heavy chain-perinatal, which are markers of myoblast proliferation and differentiation, and a strong induction of muscle regulatory factor (MRF) expression. Experiments in cultured muscle cells showed that IGF-I-induced MRF expression is facilitated by inactivation of GSK-3beta and selectively occurs in the myoblast population. This study suggests that induction of IGF-I expression and Akt phosphorylation during recovery from muscle atrophy is independent of the magnitude of muscle regrowth. Moreover, our data demonstrate for the first time that the regenerative response characterized by myoblast proliferation, differentiation, and increased MRF expression in recovering muscle is associated with the magnitude of regrowth and may be regulated by inactivation of GSK-3beta.

The cooperative transforming effects of PAX3-FKHR and IGF-II on mouse myoblasts

Alveolar rhabdomyosarcoma (ARMS) cells express high levels of PAX3-FKHR and IGF-II. In this study, we have investigated the effects of PAX3-FKHR and IGF-II on the expression of muscle regulatory factors (myf5, MyoD and myogenin), and platelet derived growth factor-B (PDGF-B) and vascular endothelial growth factor (VEGF) in mouse C2C12 myoblasts in vitro. PAX3-FKHR induced cell cycling of C2C12 cells and promoted proliferation whilst blocking myogenesis. IGF-II inhibited their differentiation without influencing proliferation. Western blotting showed that PAX3-FKHR and IGF-II blocked the expression of myogenin and MyoD respectively. Since MyoD affects early myogenesis and myogenin controls terminal differentiation, a combination of PAX3-FKHR and IGF-II synergistically blocks myogenesis at several different stages in differentiation. We have also shown that the major survival and angiogenic cytokines, PDGF-B and VEGF, were induced by IGF-II and PAX3-FKHR respectively. A combination of PAX3-FKHR and IGF-II could synergistically up regulate the expression of PDGF-B and VEGF and stabilize their high expression levels. Our results suggest that high expression of PAX3-FKHR and IGF-II in ARMS synergistically play a key role in oncogenesis and tumour progression of ARMS.

The reorganisation of constitutive heterochromatin in differentiating muscle requires HDAC activity

Constitutive heterochromatin was once thought to be remarkably stable in composition and transcriptionally inert, but has recently been shown to be surprisingly dynamic. Here, we show that terminal muscle differentiation results in a global reorganisation and spatial clustering of constitutive heterochromatin. This is accompanied by enhanced histone H3K9 and H4K20 tri-methylation across major satellite regions and increased levels of major and minor satellite-encoded transcripts. Histone deacetylase (HDAC) activity is known to be important for initiating muscle differentiation. However, here, we show that low doses of HDAC inhibitors applied after the onset of muscle differentiation prevent the spatial reorganisation of constitutive heterochromatin while allowing terminal differentiation to proceed. Under these conditions, HDAC inhibition interferes with histone methylation and blocks centromere clustering, but does not prevent the temporal expression of muscle regulatory factors or the accumulation of centromere-derived transcripts. The demonstration that HDAC activity is required for spatial relocation of centromeres in differentiating muscle provides a convenient system in which the molecular drivers of differentiation-induced chromosome repositioning can be dissected.

Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes.

Skeletal myogenesis is associated with the activation of four muscle regulatory factors (MRFs): Myf5, MyoD, Myogenin and MRF4. Here we report that p38 mitogen-activated protein kinase represses the transcriptional activity of MRF4 (involved in late stages of myogenesis), resulting in downregulation of specific muscle genes. MRF4 is phosphorylated in vitro and in vivo by p38 on two serines (Ser31 and Ser42) located in the N-terminal transactivation domain, resulting in reduced MRF4-mediated transcriptional activity. In contrast, nonphosphorylatable MRF4 mutants display increased transcriptional activity and are able to advance both myoblast fusion and differentiation. We also show that expression of desmin and alpha-actin, but not muscle creatin kinase, decreased at late stages of muscle differentiation, correlating with the induction of MRF4 and p38 activation. Accordingly, inhibition of p38 during late myogenesis results in the upregulation of both desmin and alpha-actin. We propose that repression of MRF4 activity by p38 phosphorylation may represent a new mechanism for the silencing of specific muscle genes at the terminal stages of muscle differentiation.

Early changes in rat diaphragm biology with mechanical ventilation.

To better characterize the effects of 24-hour mechanical ventilation on diaphragm, the expression of myogenic transcription factors, myosin heavy chains, and sarcoplasmic/endoplasmic reticulum calcium-ATPase pumps was examined in rats. In the diaphragm of mechanically ventilated animals, the mRNA of MyoD, myosin heavy chain-2a and -2b, and sarcoplasmic/endoplasmic reticulum calcium-ATPase-1a decreased, whereas myogenin mRNA increased. In the diaphragm of anesthetized and spontaneously breathing rats, only the mRNA of MyoD and myosin heavy chain-2a decreased. MyoD and myogenin protein expression followed the changes at the mRNA, whereas the myosin heavy chain isoforms did not change. Parallel experiments involving the gastrocnemius were performed to assess the relative contribution of muscle shortening versus immobilization-induced deconditioning on muscle regulatory factor expression. Passive shortening produced no additional effects compared with immobilization-induced deconditioning. The overall changes followed a remarkably similar pattern except for MyoD protein expression, which increased in the gastrocnemius and decreased in the diaphragm while its mRNA diminished in both muscles. The early alterations in the expression of muscle protein and regulatory factors may serve as underlying molecular basis for the impaired diaphragm function seen after 24 hours of mechanical ventilation. Whether immobilization-induced deconditioning and/or passive shortening play a role in these alterations could not be fully unraveled.

The block of ryanodine receptors selectively inhibits fetal myoblast differentiation.

Differentiation and morphogenesis of skeletal muscle are complex and asynchronous events that involve various myogenic cell populations and extracellular signals. Embryonic and fetal skeletal myoblasts are responsible for the formation of primary and secondary fibers, respectively, although the mechanism that diversifies their fate is not fully understood. Calcium transients appear to be a signaling mechanism that is widely utilized in differentiation and embryogenesis. In mature skeletal muscle, calcium transients are generated mainly by ryanodine receptors (type 1 and type 3), which are involved in excitation-contraction coupling. However, it is not clear whether the activity of these receptors is important for contractile activity alone or whether it may also play a role in regulating the differentiation/developmental processes. To clarify this point, we first examined the expression of the receptors during development. The results show that the expression of both receptors appears as early as E13 during limb muscle development and parallels the expression of skeletal myosin. The expression and the activity of both receptors is maintained in vitro by all myogenic cell populations isolated from different stages of development, including somitic, embryonic and fetal myoblasts and satellite cells. Blocking ryanodine receptor activity by using ryanodine inhibits in vitro differentiation of fetal myoblasts (judged by the expression of sarcomeric myosin and formation of multinucleated myotubes) but not of somitic or embryonic and satellite muscle cells. This block is caused by the transcriptional inhibition of markers characteristic of terminal differentiation, rather than commitment, as the expression of muscle regulatory factors is not impaired by ryanodine treatment. Taken together, the data reported in this paper demonstrate that, although calcium transients represent a general mechanism for the control of differentiation and development, multiple calcium-dependent pathways may be relevant in different myogenic populations during development. Moreover, since fetal myoblasts are responsible for the formation of secondary fibers during development, and therefore for the building of the bulk of muscular mass, these results suggest that calcium release from ryanodine receptors plays a role in the histogenesis of mammalian skeletal muscle.

THE EFFECT OF ALTERNATING PERIODS OF LOADING AND UNLOADING ON SATELLITE CELL PROLIFERATION IN ADULT QUAIL MUSCLE

This study tested the hypothesis that hypertrophic adaptation following previous loading-unloading periods doesn't require satellite cell activation but results in increased protein accumulation and expression of muscle regulatory factors. The left wing of 18 Japanese quail received a stretch overload corresponding to approximately 10% bodyweight, while the contralateral side served as an intra-animal control. The first group of birds received stretch for 14 days (S14), after which they were sacrificed. The second group underwent a 14-day unloading period after the initial 14-day stretch (U14). The third group of birds was reloaded for 7 days (R7), while the fourth group was reloaded for 14 days (R14) subsequent to the 28 days loading-unloading intervention. Muscle weight increased similarly after 14 days of weighting (142%) and after 7 (143%) or 14 (136%) days of reloading relative to the intra-animal control muscles. Experimental muscles from group U14 had atrophied relative to group S14, but they were still 44% larger than the intra-animal control. Satellite cells were labeled during initial weighting in group S14 or during reweighting in groups R7 and R14 using a constant release BrdU pellet. Nuclei that were immunopostive for BrdU were visualized by immunocytohemistry and the percent of labeling was calculated relative to the total number of nuclei in each muscle cross-section. BrdU positive nuclei (satellite cells) represented 80% of the total myonuclei pool after 14 days of loading and this was similar to the level of satellite cell activation after 7 days of reloading. These data suggest that satellite cells proliferate and are involved in hypertrophic adaptations following unloading. Funded by NIH AG17143