mRNA levels for alpha-subunit of prolyl 4-hydroxylase and fibrillar collagens in immobilized rat skeletal muscle.

There is evidence that immobilization causes a decrease in total collagen synthesis in skeletal muscle within a few days. In this study, early immobilization effects on the expression of prolyl 4-hydroxylase (PH) and the main fibrillar collagens at mRNA and protein levels were investigated in rat skeletal muscle. The right hindlimb was immobilized in full plantar flexion for 1, 3, and 7 days. Steady-state mRNAs for alpha- and beta-subunits of PH and type I and III procollagen, PH activity, and collagen content were measured in gastrocnemius and plantaris muscles. Type I and III procollagen mRNAs were also measured in soleus and tibialis anterior muscles. The mRNA level for the PH alpha-subunit decreased by 49 and 55% (P < 0.01) in gastrocnemius muscle and by 41 and 39% (P < 0.05) in plantaris muscle after immobilization for 1 and 3 days, respectively. PH activity was decreased (P < 0.05-0.01) in both muscles at days 3 and 7. The mRNA levels for type I and III procollagen were decreased by 26-56% (P < 0.05-0.001) in soleus, tibialis anterior, and plantaris muscles at day 3. The present results thus suggest that pretranslational downregulation plays a key role in fibrillar collagen synthesis in the early phase of immobilization-induced muscle atrophy.     

Gender related and dexamethasone induced differences in the mRNA levels of the MRF genes in rat anterior tibial skeletal muscle

Muscle formation and postnatal growth is under the control of the muscle regulatory factors (MRF) gene family, consisting of four genes: MyoD1, myogenin, myf-5, and myf-6. Muscle mass is also known to be affected by specific drugs, like glucocorticoids. Glucocorticoids have also been characterized as muscle atrophying agents. However, glucocorticoids are also the only drugs reported to have a beneficial effect on the treatment of muscle degenerative disorders. Since muscle mass relates to gender, this may be partially caused by gender. The aim of this study is to investigate gender-related basal and dexamethasone-induced expression of the MRF genes. Gender-specific MRF mRNA levels were investigated in anterior tibial muscles of the rat. Myogenin, myf-5, and myf-6 mRNA level was significantly higher in female rats than in male rats. Since muscle mass is usually higher in males, we conclude that the development of gender-related differences in muscle mass is not primarily under the control of the mRNA levels of the MRF genes. Male rats treated with dexamethasone for 14 days (1 mg per kg body weight) showed increased levels of MyoD1, myogenin and myf-5 compared to control male rats. Female rats treated with dexamethasone showed decreased expression of myf-6 compared to control female rats. These results suggest that dexamethasone increase satellite cell-specific MRF activity in male muscle tissue, which is suggested to be associated with muscle hypertrophy, while maintenance of muscle tissue is affected in female muscle tissue. Therefore, we conclude that both basal and dexamethasone-induced MRF gene mRNA levels are regulated gender-specific.     

Serum response factor plays an important role in the mechanically overloaded plantaris muscle of rats

Molecular signaling pathways linking the hypertrophy after mechanical overloading in vivo have not been identified. Using western blot analysis, immunoprecipitation, and immunohistochemistry, we investigated the effect of the mechanical overloading state on RhoA, serum response factor (SRF), and MyoD in the rat plantaris muscle. Adult male rats (10 weeks of age) were used in this experiment. Compensatory enlargement of the plantaris muscle was induced in one leg of each rat by surgical removal of the ipsilateral soleus and gastrocnemius muscles. In the normal plantaris muscle of rats, slight expression of RhoA and SRF was observed in the quiescent satellite cells possessing CD34 and c-Met. Western blotting using the homogenate of whole muscle clearly showed that mechanical overloading of the plantaris muscle significantly increased the amount of RhoA during 3-6 days postsurgery. Threonine phosphorylation of SRF occurred at 2-4 h after mechanical overloading. The most marked increase in SRF protein was observed in the hypertrophied muscle at 6 days postsurgery. At 2 days postoperation, SRF immunoreactivity was not detected in the proliferating satellite cells possessing bromodeoxyuridine and in the infiltrating macrophages expressing ED1 in the overloaded muscle by surgical removal. The SRF protein was colocalized with RhoA, FAK, and myogenin but not Myf-5 in many mononuclear cells at 6 days of functional overload. At this time, MyoD immunoreactivity was detected in the cytoplasm of mononuclear cells (possibly satellite cell-derived myoblasts) possessing SRF protein at the nucleus. These results suggest that the signaling pathway through RhoA-FAK-SRF is important to the differentiation of satellite cells by interacting MyoD and myogenin in the hypertrophied muscle of rats.     

An E-box within the MHC IIB gene is bound by MyoD and is required for gene expression in fast muscle

The myosin heavy chain (MHC) IIB gene is selectively expressedin skeletal muscles, imparting fast contractile kinetics. Why the MHCIIB gene product is expressed in muscles like the tibialis anterior(TA) and not expressed in muscles like the soleus is currently unclear.It is shown here that the mutation of an E-box within the MHC IIBpromoter decreased reporter gene activity in the fast-twitch TA muscle90-fold as compared with the wild-type promoter. Reporter geneexpression within the TA required this E-box for activation of aheterologous construct containing upstream regulatory regions of theMHC IIB promoter linked to the basal 70-kDa heat shock protein TATApromoter. Electrophoretic mobility shift assays demonstrated thatmutation of the E-box prevented the binding of both MyoD and myogeninto this element. In cotransfected C₂C₁₂myotubes and Hep G2 cells, MyoD preferentially activated the MHC IIBpromoter in an E-box-dependent manner, whereas myogenin activated theMHC IIB promoter to a lesser extent, and in an E-box-independent manner. A time course analysis of hindlimb suspension demonstrated thatthe unweighted soleus muscle activated expression of MyoD mRNA beforethe de novo expression of MHC IIB mRNA. These data suggest a possiblecausative role for MyoD in the observed upregulation of MHC IIB in theunweighted soleus muscle.     

Stretch-induced myogenin, MyoD, and MRF4 expression and acute hypertrophy in quail slow-tonic muscle are not dependent upon satellite cell proliferation

 The objectives of these studies were to determine if (1) hypertrophy-stimulated myogenic regulatory factor (MRF) mRNA increases occur in the absense of proliferating satellite cells, and (2) acute hypertrophy occurs without satellite cell proliferation. Adult and aged quails were exposed to 0 or 2500 Rads gamma irradiation, and then wing muscles were stretch-overloaded for 3 or 7 days. MRF mRNA levels in stretch-overloaded and contralateral anterior latissimus dorsi (ALD) muscles were determined after 3 days; hypertrophy was determined after 7 days. The elimination of proliferating cells in irradiated muscles was verified histologically by bromodeoxyuridine incorporation. Relative levels of MRF4, MyoD, and myogenin mRNA were elevated 100%–400% in stretch-overloaded ALD muscles from irradiated adult quails indicating that satellite cell proliferation was not a prerequisite for MRF mRNA increases. Myogenin was the only MRF that exhibited mRNA increases that were lowered by irradiation. This suggests that satellite cells contribute only to myogenin mRNA increases in non-irradiated adult muscles following 3 days of stretch-overload. Stretch-overloaded ALD muscles from aged quails had a relative increase in myogenin mRNA of ∼150%. The myogenin increase was the same in non-irradiated and irradiated aged animals and also the same as that in stretch-overloaded muscles from irradiated adult quails. Together, these data indicate that attenuated increases in MRF expression in muscles from aged animals are attributable to lower satellite cell MRF expression. ALD muscle masses and protein contents in adult irradiated quails approximately doubled after 7 days of stretch-overload demonstrating hypertrophy despite the elimination of satellite cell proliferation. Received: 5 June 1998 / Accepted: 19 November 1998     

Expression of myogenic factors in denervated chicken breast muscle: isolation of the chicken Myf5 gene.

In this study, we have isolated and characterized the chicken Myf5 gene, and cDNA clones encoding chicken MyoD1 and myogenin. The chicken Myf5 and MRF4 genes are tandemly located on a single genomic DNA fragment, and the chicken Myf5 gene is organized into at least three exons. Using genomic and cDNA probes, we further analyzed the mRNA levels of four myogenic factors during chicken breast muscle development. This analysis revealed that myogenin expression is restricted to in ovo stages in breast muscle, and is not detectable in neonatal and adult stages. On the other hand, Myf5 expression is detectable until day 7 post-hatching, and is not found in adult muscle, whereas high levels of MyoD1 and MRF4 are detectable at all stages. To further understand the roles of innervation on muscle maturation, we analyzed the expression of the four myogenic factors in denervated adult breast muscle. We found that MyoD1, myogenin, and MRF4 are induced at high levels in denervated muscle, whereas no change occurs in the level of Myf5. These studies suggest that innervation controls the relative abundance and type of myogenic factors that are expressed in adult muscle, and that when nerve control is removed, the muscle reverts to a neonatal phenotype, with the enhanced expression of three myogenic factors (MyoD1, myogenin, and MRF4).     

Maternal protein restriction induce skeletal muscle changes without altering the MRFs MyoD and myogenin expression in offspring

Stimuli during pregnancy, such as protein restriction, can affect morphophysiological parameters in the offspring with consequences in adulthood. The phenomenon known as fetal programming can cause short- and long-term changes in the skeletal muscle phenotype. We investigated the morphology and the myogenic regulatory factors (MRFs) MyoD and myogenin expression in soleus, SOL; oxidative and slow twitching and in extensor digitorum longus, EDL; glycolytic and fast twitching muscles in the offspring of dams subjected to protein restriction during pregnancy. Four groups of male Wistar offspring rats were studied. Offspring from dams fed a low-protein diet (6?% protein, LP) and normal protein diet (17?% protein, NP) were euthanized at 30 and 112?days old, and their muscles were removed and kept at ?80?°C. Muscles histological sections (8?μm) were submitted to a myofibrillar adenosine triphosphatase histochemistry reaction for morphometric analysis. Gene and protein expression levels of MyoD and myogenin were determined by RT-qPCR and western blotting. The major findings observed were distinct patterns of morphological changes in SOL and EDL muscles in LP offspring at 30 and 112?days old without changes in MRFs MyoD and myogenin expression. Our results indicate that maternal protein restriction followed by normal diet after birth induced morphological changes in muscles with distinct morphofunctional characteristics over the long term, but did not alter the MRFs MyoD and myogenin expression. Further studies are necessary to better understand the mechanisms underlying the maternal protein restriction response on skeletal muscle.     

Related expression of MyoD and Myf5 with myosin heavy chain isoform types in bovine adult skeletal muscles

Skeletal muscles are characterized as fast and slow muscles, according to the expression pattern of myosin heavy chain (MyHC) isoforms in the muscle fibers. To investigate the relationships between MyHC isoforms and myogenic regulatory factors (MRFs) including MyoD, Myf5, myogenin, and MRF4 in adult skeletal muscles, expressions of these MRFs in the ten muscles of three cows were analyzed by a semi-quantitative RT-PCR. The results showed that MyoD expression was significantly lower in the lingual muscles (TN), masseter (MS) and diaphragm (DP), which lack MyHC-2x (fast glycolytic) expression and abound with MyHC-slow (slow oxidative) and/or MyHC-2a (fast oxidative), than it was in the pectoralis (PP), psoas major (PM), longissimus thoracis (LT), spinnalis (SP), semitendinosus (ST), semimembranosus (SM), and biceps femoris (BF). In contrast, the Myf5 expression in TN, MS, and DP was significantly higher than in PM, LT, ST, SM, and BF. No significant difference was observed in myogenin and MRF4 expression among the muscles tested. The results suggest that MyoD and Myf5 influence the MyHC isoform expression, although the effects are not decisive in specifying the phenotypes of adult muscles.     

Satellite cell proliferation is reduced in muscles of obese Zucker rats but restored with loading

The obese Zucker rat (OZR) is a model of metabolic syndrome, which has lower skeletal muscle size than the lean Zucker rat (LZR). Because satellite cells are essential for postnatal muscle growth, this study was designed to determine whether reduced satellite cell proliferation contributes to reduced skeletal mass in OZR vs. LZR. Satellite cell proliferation was determined by a constant-release 5-bromo-2-deoxyuridine (BrdU) pellet that was placed subcutaneously in each animal. Satellite cell proliferation, as determined by BrdU incorporation, was significantly attenuated in control soleus and plantaris muscles of the OZR compared with that shown in the LZR. To determine whether this attenuation of satellite cell activity could be rescued in OZR muscles, soleus and gastrocnemius muscles were denervated, placing a compensatory load on the plantaris muscle. In the LZR and the OZR after 21 days of loading, increases of approximately 25% and approximately 30%, respectively, were shown in plantaris muscle wet weight compared with that shown in the contralateral control muscle. The number of BrdU-positive nuclei increased similarly in loaded plantaris muscles from LZR and OZR. Myogenin, MyoD, and Akt protein expressions were lower in control muscles of OZR than in those of the LZR, but they were all elevated to similar levels in the loaded plantaris muscles of OZR and LZR. These data indicate that metabolic syndrome may reduce satellite cell proliferation, and this may be a factor that contributes to the reduced mass in control muscles of OZR; however, satellite cell proliferation can be restored with compensatory loading in OZR.