Expression Of Myogenic Regulatory Factors Research Articles

Altered Expression of Myogenic Regulatory Factors in the Mouse Model of Amyotrophic Lateral Sclerosis

Background: In the superoxide dismutase 1 (SOD1)-G93A mouse model of amyotrophic lateral sclerosis (ALS), skeletal muscle is a key target of mutant SOD1 toxicity. However, the expression of factors that control the regenerative potential of the muscle is unknown in this model. Objective: To characterize the expression of satellite cell marker Pax7 and myogenic regulatory factors (MRF) in skeletal muscle of SOD1-G93A mice at different stages of the disease. Methods: The expressions of Pax7, Myod1, Myf5 and myogenin (Myog) were determined by quantitative real-time PCR and by Western blotting from the grouped gastrocnemius, quadriceps and soleus muscles of SOD1-G93A mice at presymptomatic, symptomatic and terminal stages of the disease, and from surgically denervated wild-type gastrocnemius muscles. Results:Pax7 mRNA and MYF5 protein were upregulated in presymptomatic mice, coinciding with increased muscle damage marker Rrad and chemokine Ccl5. All MRF transcripts and most proteins (excluding MYOG) were increased, starting from 3 months of age, simultaneously with increased expression of denervation marker Chrna1. However, in the terminal stage, no protein increase was evident for Pax7 or any of the MRF despite the increased mRNA levels. The transcripts for chemokine Ccl2 and chemokine receptor Cxcr4 were increased starting from the onset of symptoms. Conclusions: The characterization of Pax7 and MRF in SOD1-G93A mice reveals a progressive induction of the myogenic program at the RNA level, but a blunted protein level response at late stages of the disease. Altered posttranscriptional and posttranslational mechanisms likely to operate, as well as the potential role of chemokine signaling in mutant SOD1 muscle, are discussed.

Role of endogenous TGF‐β family in myogenic differentiation of C2C12 cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis.

Blocking the Myostatin Signal With a Dominant Negative Receptor Improves the Success of Human Myoblast Transplantation in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Myostatin (MSTN) is a negative regulator of skeletal muscle development and responsible for limiting regeneration. It binds with high affinity to the activin type IIB receptor (ActRIIB). Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the MSTN signal with expression of a dominant negative mutant of ActRIIB (dnActRIIB). In vitro, blocking MSTN activity with a lentivirus carrying dnActRIIB increased proliferation and fusion of human myoblasts because MSTN regulates the expression of several myogenic regulatory factors. In vivo, myoblasts infected with the dnActRIIB lentivirus were transplanted in immunodeficient dystrophic mice. Dystrophin immunostaining of tibialis anterior (TA) cross-sections of these mice 1 month post-transplantation revealed more human dystrophin-positive myofibers following the transplantation of dnActRIIB myoblasts than of control myoblasts. Thus, blocking the MSTN signal with dnActRIIB improved the success of myoblast transplantation by increasing the myoblast proliferation and fusion and changed the expression of myogenic regulatory factors.

The expression of myogenic regulatory factors and muscle growth factors in the masticatory muscles of dystrophin-deficient (mdx) mice.

The activities of myogenic regulatory factors (MRF) and muscle growth factors increase in muscle that is undergoing regeneration, and may correspond to some specific changes. Little is known about the role of MRFs in masticatory muscles in mdx mice (the model of Duchenne muscular dystrophy) and particularly about their mRNA expression during the process of muscle regeneration. Using Taqman RT-PCR, we examined the mRNA expression of the MRFs myogenin and MyoD1 (myogenic differentiation 1), and of the muscle growth factors myostatin, IGF1 (insulin-like growth factor) and MGF (mechanogrowth factor) in the masseter, temporal and tongue masticatory muscles of mdx mice (n = 6 to 10 per group). The myogenin mRNA expression in the mdx masseter and temporal muscle was found to have increased (P < 0.05), whereas the myostatin mRNA expressions in the mdx masseter (P < 0.005) and tongue (P < 0.05) were found to have diminished compared to those for the controls. The IGF and MGF mRNA amounts in the mdx mice remained unchanged. Inside the mdx animal group, gender-related differences in the mRNA expressions were also found. A higher mRNA expression of myogenin and MyoD1 in the mdx massterer and temporal muscles was found in females in comparison to males, and the level of myostatin was higher in the masseter and tongue muscle (P < 0.001 for all comparisons). Similar gender-related differences were also found within the control groups. This study reveals the intermuscular differences in the mRNA expression pattern of myogenin and myostatin in mdx mice. The existence of these differences implies that dystrophinopathy affects the skeletal muscles differentially. The finding of gender-related differences in the mRNA expression of the examined factors may indicate the importance of hormonal influences on muscle regeneration.

Nutritional Restriction Prior to Conception in the Sow Alters Embryonic Development and Candidate Gene Expression in the Placenta and Endometrium.

The extent to which maternal nutrition alters placental and fetal development through epigenetic mechanisms, is still uncertain. In an established experimental model using the catabolic primiparous sow, feed restriction during lactation affects embryonic survival and development during the subsequent gestation. In a continuation of these studies, primiparous sows (n=34) were either fully fed (Control:C) or restricted to 60% of voluntary feed intake (Restrict:R) during the third week of a 21-d lactation and re-bred after weaning. Sows were euthanized at d 30 of gestation and reproductive and litter characteristics were recorded. Embryos and placentae (n=488 of each) were collected for each sow, and a sample of endometrium (n=68) was taken from the left and right horn from each sow, frozen immediately in liquid nitrogen and stored at -80°C. Total RNA was extracted from placental, endometrial and embryonic samples collected, DNAse-treated, reverse transcribed, and real-time PCR was used to determine transcript abundance of candidate genes involved in regulating endometrial and placental vascularity and embryonic muscle development. The relative deltaCt values were calculated using cyclophilin as an internal control, and data were analyzed using mixed models analysis with treatment and sex of embryo as independent variables and sow as a random variable. There were no differences in ovulation rate, embryonic survival or number of embryos in utero between C and R sows. However, litter sex ratio was different with more males in the R sows (P<0.05) and embryo weight was reduced (P<0.05) in R compared to C sows. Embryo weight and litter sex ratio were also related to sow weight loss in the previous lactation. When genes regulating vascular development were considered, FGF2 and VEGFA mRNA expression was lower in the endometrium, while ANGPT2 was lower (P<0.05) in the placenta, recovered from R sows. Expression of myogenic regulatory factors analyzed (MYF5, MYF6, MYOG, MYOD1, PAX3, GLI1 and WNT10b) was not different between C and R sows at d 30 of gestation. In addition, the expression of some imprinted genes were examined, and IGF2 expression tended to be higher (P=0.059) and INSR expression was higher (P=0.049) in the R embryos, and IGF2R demonstrated a tendency for a sex by treatment interaction (P=0.066) in the placenta. These results confirm that maternal metabolic state prior to conception can adversely affect embryonic size and sex ratio in the next litter. These effects involved expression of genes related to vascular development in the uterus and placenta, and were consistent with the concept of epigenetic regulation of developmentally important genes. (platform)

Low-magnitude vertical vibration enhances myotube formation in C2C12 myoblasts

Whole body vibration training is widely used in rehabilitation and sports activities to improve muscle strength, balance, and flexibility. However, the molecular mechanisms of vertical vibration (VV) training and their effect on the myogenesis of myoblasts remain undefined. This study was undertaken to address the hypothesis that VV can enhance the expression of ECM proteins and myogenic regulatory factors (MRFs) in myoblasts and, in turn, increase myotube formation. Using real-time PCR, Western blot analysis, and immunofluorescence studies, we examined the effect of VV treatment with frequencies of 5, 8, or 10 Hz on the expression of ECM proteins and MRFs as well as myotube formation in C2C12 myoblasts. We showed that VV stimulation is safe and effective at stimulating myogenesis in C2C12 myoblasts. The levels of expression of the ECM proteins type I collagen and decorin were the highest after VV treatment at frequencies of 8 and 10 Hz. Expression of the MRFs MyoD and myogenin increased after VV stimulation in a time- and dose-dependent manner. The total number of myotubes formed, as well as the length and the average area of myotubes, were substantially increased following VV treatment at frequencies of 8 to 10 Hz. In conclusion, VV treatment at frequencies of 8 to 10 Hz can stimulate the expression of ECM proteins and MRFs in myoblasts and, in turn, increase myotube formation.

Quantitative expression of myogenic regulatory factors MyoD and myogenin in pacu ( Piaractus mesopotamicus) skeletal muscle during growth

Skeletal muscle growth is regulated by differential expression of myogenic regulatory factors (MRFs). We evaluated hyperplasia, hypertrophy and quantitative expression of MRFs MyoD and myogenin in 45, 90, 180, and 400 days post-hatching (dph) and adult pacu ( Piaractus mesopotamicus) skeletal muscle. Transverse sections of white dorsal muscles were obtained to evaluate hypertrophy and hyperplasia. MyoD and myogenin gene expression was determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Pacu skeletal muscle had similar morphology at all stages. The highest and the lowest frequencies of fiber diameters <20 μm were found at the 45 dph and adult stages, respectively. Their frequency was similar in the 90, 180, and 400 dph stages. The highest percentage of >50 μm diameter fibers were found in 180 and 400 dph, and adult fish. Hyperplasia was the main mechanism observed in pacu skeletal muscle growth at 45 dph; this declined through 90, 180, and 400 dph and remained low in adult fish; the latter presented hypertrophy as the main mechanism responsible for skeletal muscle growth. The high frequencies of 20–50 μm diameter fibers at 90, 180, and 400 dph can be related to intense hypertrophy. The mRNA levels for MyoD and myogenin were similar in 45, 90, and 400 dph and adult fish, peaking at 180 dph. The high MyoD expression at 180 dph can be related to intense myoblast proliferation and hyperplasia, while high myogenin expression can be related to intense myoblast differentiation and fusion during hypertrophy. MyoD and myogenin expression patterns in adults can respectively be associated with myoblast proliferation and differentiation, which both contribute to hypertrophy. Differential MyoD and myogenin expression in pacu white muscle probably is associated with differences in growth patterns during the stages analyzed. In this study, the 180 dph pacu could represent an interesting phase to investigate suitable strategies in commercial fish production focusing on skeletal muscle growth improvement to raise healthy, fast-growing fish.

Muscle stem cells in developmental and regenerative myogenesis

Skeletal muscle development serves as a paradigm for cell lineage specification and cell differentiation. Adult skeletal muscle has high regenerative capacity, with satellite cells the primary source of this capability. The present review describes recent findings on developmental and adult myogenesis with emphasis on emerging distinctions between various muscle groups and stages of myogenesis. Muscle progenitors of the body are derived from multipotent cells of the dermomyotome and express the transcription factors Pax3 and Pax7. These cells self-renew or induce expression of myogenic regulatory factors (MRFs) and differentiate. The roles of Pax3, Pax7 and specific myogenic regulatory factor progenitor populations in trunk and limb myogenesis have been identified through cell ablation in the mouse. Various head muscles and associated satellite cells have differing developmental origins, and rely on distinct combinations of transcriptional regulators, than trunk and limb muscles. Several genetic and sorting protocols demonstrate that satellite cells are heterogeneous with some possessing stem cell properties; the relative roles of lineage and niche in these properties are being explored. Although cellular mechanisms of developmental, postnatal and adult regenerative myogenesis are thought to be similar, recent studies reveal distinct genetic requirements for embryonic, fetal, postnatal and adult regenerative myogenesis. Genetic determinants of formation or repair of various muscles during different stages of myogenesis are unexpectedly diverse. Future studies should illuminate these differences, as well as mechanisms that underlie stem cell properties of satellite cells.

Exercise Training Increases LC3, Marker Of Autophagy

Microtubule associated protein 1A/1B-Light Chain 3(LC3) become known serve as a marker for autophagy. Autophagy or cell death type II, is responsible for the self-degradative processes implicated in the normal turnover of cellular components as well as in response to such stress as nutrient starvation and/or growth factor deprivation. One of the functions of autophagy is to remove damaged organelles in order to prevent further injury and cellular dysfunction. However, it remains unknown how LC3 would be changed by exercise training. PURPOSE: To investigate the effect of exercise training on apoptosis, autophagy and myogenic regulatory factors in the gastrocnemius muscle in 9 month aged mice. METHODS: Sixteen of male ICR mice were randomly assigned into control group (CON) and treadmill exercise training group (EX). EX were run on treadmill at 16.3m/min for 40min on 3% slope 5d/wk for 8 wks. Muscle samples were taken from the gastrocnemius at 48h after the last training session. Gastrocnemius muscle was weighted and protein level of LC3 and caspase-3 were measured by western blot analysis. Real-time PCR analysis was performed on the following genes: MyoD, Myf5, Myogenin, MAFbx (Muscle atrophy F-box). 18s was used as the housekeeping gene. RESULTS: Cleaved LC3 and caspase-3 were increased in EX compared to CON. Gene expression of myogenic regulatory factors was not significantly different between CON and EX. Moreover, gene expression of MAFbx was not different between EX and CON. However, gastrocnemius muscle wt-to-body wt ratio was significant increased in EX group (0.502±0.029) compared with CON (0.322±0.068). CONCLUSION: Our results demonstrated that protein expression of cleaved form of LC3 as well as caspase-3 increased by the exercise training. However, there were no concomitant changes in myogenic regulatory factors and MAFbx with exercise training in ICR mice skeletal muscle. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology(2009-0077015).

Skeletal muscle cellularity and expression of myogenic regulatory factors and myosin heavy chains in rainbow trout (Oncorhynchus mykiss): Effects of changes in dietary plant protein sources and amino acid profiles

The nutritional regulation of skeletal muscle growth is very little documented in fish. The aim of the study presented here was to determine how changes in dietary plant protein sources and amino acid profiles affect the muscle growth processes of fish. Juvenile rainbow trout (Oncorhynchys mykiss) were fed two diets containing fish meal and a mixture of plant protein sources either low (control diet) or rich in soybean meal (diet S). Both diets were supplemented with crystalline indispensable amino acids (IAA) to match the rainbow trout muscle IAA profile. Diet S was also supplemented with glutamic acid, an AA present in high quantities in trout muscle. Rainbow trout fed diets C and S were not significantly different in terms of overall somatic growth or daily nitrogen gain, although their parameters of dietary protein utilisation differed. Distribution of skeletal white muscle fibre diameter and expression of certain selected muscle genes were also affected by dietary changes. In the white muscle, diet S led to a significant decrease (×0.9) in the mean and median diameters of muscle fibres, to a significant decrease (×0.6) in the expression of MyoD and to a significant increase (×1.7) in the expression of fast-MHC, with no significant changes in myogenin expression. There was no change in the expression of the genes analysed in lateral red muscle (MyoD, MyoD2, myogenin and slow-MHC). These results demonstrated that changes occurred in skeletal white muscle cellularity and expression of MyoD and fast-MHC, although overall growth and protein accretion were not modified, when a diet rich in soybean meal and glutamic acid was ingested. Present findings also indicated that the white and red muscles of rainbow trout are differently affected by nutritional changes.

Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells

The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18 degrees C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression (R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoD1a transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoD1a and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the G(1) and S/G(2) phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in the Atlantic salmon lineage.

Quantitative comparison of the expression of myogenic regulatory factors in flounder (Paralichthys olivaceus) embryos and adult tissues

MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal differentiation and lineage maintenance. To better understand the function of myogenic regulatory factors in muscle development of flounder, an important economic fish in Asia, real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD, Myf5, and myogenin at early stages of embryo development, and in different tissues of the adult flounder. The results show that, Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions of Myf5, yoD, and myogenin at the early stages have a common characteristic: expression gradually increased to a peak level, and then gradually decreased to an extremely low level. In the adult flounder, the expression of the three genes in muscle is much higher than that in other tissues, indicating that they are important for muscle growth and maintenance of grown fish. During embryonic stages, the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation. When the MyoD expression is over 30% of its highest level, the muscle cells enter the differentiation stage.

Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

Satellite cells/myoblasts account for the majority of muscle regenerative potential in response to injury and muscular adaptation to exercise. Although the ability to influence this process would provide valuable benefits for treating a variety of patients suffering from muscle loss, the regulatory mechanisms of myogenesis are not completely understood. We have tested the hypothesis that transforming growth factor-beta-activated kinase 1 (TAK1) is an important regulator of skeletal muscle formation. TAK1 is expressed in proliferating C2C12 myoblasts, and its levels are reduced upon differentiation of myoblasts into myotubes. In vivo, TAK1 is predominantly expressed in developing skeletal muscle of young mice. However, the expression of TAK1 was significantly up-regulated in regenerating skeletal muscle of adult mice. Overexpression of a dominant negative mutant of TAK1 or knockdown of TAK1 inhibited the proliferation and differentiation of C2C12 myoblasts. TAK1 was required for the expression of myogenic regulatory factors in differentiating myoblasts. Genetic ablation of TAK1 also inhibited the MyoD-driven transformation of mouse embryonic fibroblasts into myotubes. Inhibition of TAK1 suppressed the differentiation-associated activation of p38 mitogen-activated protein kinase (MAPK) and Akt kinase. Overexpression of a constitutively active mutant of MAPK kinase 6 (MKK6, an upstream activator of p38 MAPK) but not constitutive active Akt restored the myogenic differentiation in TAK1-deficient mouse embryonic fibroblasts. Insulin growth factor 1-induced myogenic differentiation was also found to involve TAK1. Collectively, our results suggest that TAK1 is an important upstream regulator of skeletal muscle cell differentiation.

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type-specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin-deficient double-muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse-transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC-2x and -2a and less -slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster-type muscles and on MyoD expression in slower-type muscles, suggesting a possible muscle type-specific effect of myostatin in skeletal muscle growth and maintenance.

Effects of Different Intensities of Resistance Exercise on Regulators of Myogenesis

A single bout of high-intensity resistance exercise is capable of activating the expression of various genes in skeletal muscle involved in hypertrophy such as myosin heavy chain (MHC) isoforms, myogenic regulatory factors (MRFs), and growth factors. However, the specific role exercise intensity plays on the expression of these genes is not well defined. The purpose of this study was to investigate the effects of exercise intensity on MHC (type I, IIA, IIX), MRF (Myo-D, myogenin, MRF-4, myf5), and growth factor (insulin-like growth factor [IGF]-1, IGF-1 receptor [IGF-R1], mechano-growth factor [MGF]) mRNA expression. Thirteen male participants (21.5 +/- 2.9 years, 86.1 +/- 19.5 kg, 69.7 +/- 2.7 in.) completed bouts of resistance exercise involving 4 sets of 18-20 repetitions with 60-65% 1 repetition maximum (1RM) and 4 sets of 8-10 repetitions with 80-85% 1RM. Vastus lateralis biopsies were obtained immediately before exercise, and at 30 minutes, 2 hours, and 6 hours after each bout. The levels of mRNA expression were determined using real-time polymerase chain reaction. Data were analyzed using 2 x 4 multivariate analysis of variance (p <or= 0.05). For both intensities, MHC type IIX, IGF-1, IGF-R1, MGF, Myo-D, myogenin, MRF-4, and myf5 mRNA were all significantly increased in response to resistance exercise by 2 hours after exercise, whereas myostatin and the cyclin-dependent kinase inhibitor p27(kip) were decreased at 2 hours after exercise (p < 0.05). Resistance exercise between 60-85% 1RM upregulates the mRNA expression of MHC and factors involved in myogenic activation of satellite cells while concomitantly decreasing expression of myogenic inhibitors.

Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts

It is known that the MyoD family members (MyoD, Myf5, myogenin, and MRF4) play a pivotal role in the complex mechanism of skeletal muscle cell differentiation. However, fragmentary information on transcription factor-specific regulation is available and data on their post-transcriptional and post-translational behavior are still missing. In this work, we combined mRNA and protein expression analysis with their subcellular localization. Each myogenic regulator factor (MRF) revealed a specific mRNA trend and a protein quantitative analysis not overlapping, suggesting the presence of post-transcriptional mechanisms. In addition, each MRF showed a specific behavior in situ, characterized by a differentiation stage-dependent localization suggestive of a post-translational regulation also. Consistently with their transcriptional activity, immunogold electron microscopy data revealed MRFs distribution in interchromatin domains. Our results showed a MyoD and Myf5 contrasting expression profile in proliferating myoblasts, as well as myogenin and MRF4 opposite distribution in the terminally differentiated myotubes. Interestingly, MRFs expression and subcellular localization analysis during C2C12 cell differentiation stages showed two main MRFs regulation mechanisms: (i) the protein half-life regulation to modulate the differentiation stage-dependent transcriptional activity and (ii) the cytoplasmic retention, as a translocation process, to inhibit the transcriptional activity. Therefore, our results exhibit that MRFs nucleo-cytoplasmic trafficking is involved in muscle differentiation and suggest that, besides the MRFs expression level, also MRFs subcellular localization, related to their functional activity, plays a key role as a regulatory step in transcriptional control mechanisms.

Lack of Effects of Creatine on the Regeneration of Soleus Muscle after Injury in Rats

Creatine (Cr) supplementation may improve muscle functional capacity in patients with neuromuscular diseases, disuse atrophy, or muscular dystrophies. Activation of myogenic satellite cells has been reported to be enhanced by Cr both in vitro and in vivo. Therefore, we hypothesized that Cr supplementation may improve the early steps of regeneration after muscle injury and may accelerate the recovery of both muscle mass and phenotype. Degeneration of left soleus muscle was induced by notexin injection in rats supplemented or not with Cr. The mass of regenerated muscles was compared with contralateral intact muscles at days 1, 3, 7, 14, 21, 28, 35, and 42 after injury. We also studied protein levels of the proliferator cell nuclear antigen (PCNA) as a marker of cell proliferation, expression of myogenic regulatory factors (MRF) as a marker of differentiation, and the myosin heavy chain (MHC) profile and activities of citrate synthase (CS) and lactate dehydrogenase (LDH) isozymes as markers of muscle phenotype maturation. Cr supplementation accelerated the recovery of muscle Cr content during the regeneration phase. Although there were no other differences between Cr-treated and nontreated rats, we observed that 1) regenerated muscle mass remained lower than that in intact muscle mass 42 d after injury, 2) PCNA and MRF expression strongly increased in regenerated muscles, 3) the MHC profile of regenerated muscles was recovered 28 d after injury, and 4) CS activity was fully recovered from day 14, whereas the specific H isozyme of lactate dehydrogenase activity remained lower than that in intact muscles until 42 d. In contrast with results from in vitro studies, Cr supplementation had no effects in vivo on the time course of recovery of rat skeletal muscle mass and phenotype after notexin-induced injury.

Phasing of muscle gene expression with fasting-induced recovery growth in Atlantic salmon.

BackgroundMany fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.ResultsHierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.ConclusionEarly responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.

G.P.16.01 Expression of myogenic regulatory factors and myo-regeneration in inflammatory myopathies

G.P.16.01 Expression of myogenic regulatory factors and myo-regeneration in inflammatory myopathies

Coordinated patterns of gene expressions for adult muscle build-up in transgenic mice expressing myostatin propeptide

BackgroundSkeletal muscle growth and maintenance are essential for human health. One of the muscle regulatory genes, namely myostatin, a member of transforming growth factor-β, plays a dominant role in the genetic control of muscle mass. Myostatin is synthesized as a precursor protein, which generates the N-terminal propeptide and the C-terminal mature myostatin peptide by a post-translational cleavage event. Previously, transgenic over-expression of myostatin propeptide in skeletal muscle results in significant muscle growth in early stages of development. The objectives of present study were to further characterize muscle growth in later stages of life and to identify genes and their expression patterns that are responsible for adult muscle build-up by myostatin propeptide.ResultsImmunohistochemical staining with an antibody to the N-terminus indicates a high level of myostatin propeptide present in the muscles of transgenic mice while there were no apparent differences in myostatin protein distribution in the muscle fibers between the transgenic and wild-type mice. Main individual muscles increased by 76–152% in the transgenic mice over their wild-type littermate mice at 12 months of age. A large number of nuclei were localized in the central and basal lamina of the myofibers in the transgenic mice as the number of nuclei per fiber and 100 μm2 area was significantly higher in transgenic mice than wild-type mice. By systemic comparisons of global mRNA expression patterns between transgenic mice and wild-type littermates using microarray and qRT-PCR techniques, we have identified distinct gene expression patterns to support adult muscle build-up by myostatin propeptide, which are comprised of enhanced expressions of myogenic regulatory factors and extracelullar matrix components, and differentially down-regulated expressions of genes related to protein degradation and mitochondrial ATP synthesis.ConclusionThe results present a coordinated pattern of gene expressions for reduced energy utilization during muscle build-up in adult stage. Enhanced muscle buildup by myostatin propeptide is sustained by reduced ATP synthesis as a result of a decreased activity of protein degradation. Myostatin propeptide may have a therapeutic application to the treatment of clinical muscle wasting problems by depressing myostatin activity.