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).     

Specific activation of the acetylcholine receptor subunit genes by MyoD family proteins

Whether the myogenic regulatory factors (MRFs) of the MyoD family can discriminate among the muscle gene targets for the proper and reproducible formation of skeletal muscle is a recurrent question. We have previously shown that, in Xenopus laevis, myogenin specifically transactivated muscle structural genes in vivo. In the present study, we used the Xenopus model to examine the role of XMyoD, XMyf5, and XMRF4 for the transactivation of the (nicotinic acetylcholine receptor) nAChR genes in vivo. During early Xenopus development, the expression patterns of nAChR subunit genes proved to be correlated with the expression patterns of the MRFs. We show that XMyf5 specifically induced the expression of the delta-subunit gene in cap animal assays and in endoderm cells of Xenopus embryos but was unable to activate the expression of the gamma-subunit gene. In embryos, overexpression of a dominant-negative XMyf5 variant led to the repression of delta-but not gamma-subunit gene expression. Conversely, XMyoD and XMRF4 activated gamma-subunit gene expression but were unable to activate delta-subunit gene expression. Finally, all MRFs induced expression of the alpha-subunit gene. These findings strengthen the concept that one MRF can specifically control a subset of muscle genes that cannot be activated by the other MRFs.     

Nerve activity-independent regulation of skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei

Electrical activity is thought to be the primary neural stimulus regulating muscle mass, expression of myogenic regulatory factor genes, and cellular activity within skeletal muscle. However, the relative contribution of neural influences that are activity-dependent and -independent in modulating these characteristics is unclear. Comparisons of denervation (no neural influence) and spinal cord isolation (SI, neural influence with minimal activity) after 3, 14, and 28 days of treatment were used to demonstrate whether there are neural influences on muscle that are activity independent. Furthermore, the effects of these manipulations were compared for a fast ankle extensor (medial gastrocnemius) and a fast ankle flexor (tibialis anterior). The mass of both muscles plateaued at approximately 60% of control 2 wk after SI, whereas both muscles progressively atrophied to <25% of initial mass at this same time point after denervation. A rapid increase in myogenin and, to a lesser extent, MyoD mRNAs and proteins was observed in denervated and SI muscles: at the later time points, these myogenic regulatory factors remained elevated in denervated, but not in SI, muscles. This widespread neural activity-independent influence on MyoD and myogenin expression was observed in myonuclei and satellite cells and was not specific for fast or slow fiber phenotypes. Mitotic activity of satellite and connective tissue cells also was consistently lower in SI than in denervated muscles. These results demonstrate a neural effect independent of electrical activity that 1) helps preserve muscle mass, 2) regulates muscle-specific genes, and 3) potentially spares the satellite cell pool in inactive muscles.     

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.     

The adaptive response of MyoD family proteins in overloaded, regenerating and denervated rat muscles.

Using Western blot analysis, we investigated whether the amount of myogenic regulatory factors differs in slow-type and fast-type muscles. In addition, we examined the adaptive response of myogenic regulatory factor protein in the overloaded rat muscles by the ablation of synergists, in the regenerating muscles following bupivacaine injection and in the denervated muscle. The amount of myogenin protein in the slow-type muscle was markedly greater. In contrast, the proteins MyoD and Myf-5 were selectively accumulated in the fast-type muscles. A gradual down-regulation of MyoD and Myf-5 proteins was detected in the denervated fast-type muscles, but not in the myogenin protein content. A rapid down-regulation of myogenic regulatory factor protein was observed both of the mechanically overloaded and in the regenerating muscles. These results indicate that the fast-type-specific gene expression in muscle is modulated by MyoD and Myf-5 proteins and suggest that myogenin protein plays an important role in the reconstruction of damaged neuromuscular connections.     

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