Myogenin modulates exercise endurance by altering skeletal muscle metabolism

The function of myogenic regulatory factors (MRFs) during adult life is not well understood. The requirement of one of these MRFs, myogenin (Myog), during embryonic muscle development suggests an equally important role in adult muscle. In this study, we have determined the function of myogenin during adult life using a conditional allele of Myog. In contrast to embryonic development, myogenin is not required for adult viability, and Myog-deleted mice exhibited no remarkable phenotypic changes during sedentary life. Remarkably, sedentary Myog-deleted mice demonstrated enhanced exercise endurance during involuntary treadmill running. Altered blood glucose and lactate levels in sedentary Myog-deleted mice after exhaustion suggest an enhanced glycolytic metabolism and an ability to excessively deplete muscle and liver glycogen stores. Traditional changes associated with enhanced exercise endurance, such as fiber type switching, and increased oxidative potential, were not detected in sedentary Myog-deleted mice. After long-term voluntary exercise, trained Myog-deleted mice demonstrated an enhanced adaptive response to exercise. Trained Myog-deleted mice exhibited superior exercise endurance associated with an increased proportion of slow-twitch fibers and increased oxidative capacity. In a parallel experiment, dystrophin-deficient young adult mice showed attenuated muscle fatigue following the deletion of Myog. These results demonstrate a novel and unexpected role for myogenin in modulating skeletal muscle metabolism.

ELUCIDATING FUNCTIONAL ROLES FOR MYOGENIN IN ADULT SKELETAL MUSCLE METABOLISM, EXERCISE CAPACITY, AND REGENERATION

The four basic helix-loop-helix myogenic transcription factors, myogenin, Myf5, MRF4, and MyoD are critical for embryonic skeletal muscle development. Myogenin is necessary for the terminal differentiation of myoblasts into myofibers during embryogenesis, but little is known about the roles played by myogenin in adult skeletal muscle function and metabolism. Furthermore, while metabolism is a well-studied physiological process, how it is regulated at the transcriptional level remains poorly understood. In this study, my aim was to determine the function of myogenin in adult skeletal muscle metabolism, exercise capacity, and regeneration. To investigate this, I utilized a mouse strain harboring the Myogflox allele and a Cre recombinase transgene, enabling the efficient deletion of myogenin in the adult mouse. Myogflox/flox mice were stressed physically through involuntary treadmill running and by breeding them with a strain harboring the Duchenne’s muscular dystrophy (DMDmdx) allele. Surprisingly, Myog-deleted animals exhibited an enhanced capacity for exercise, running farther and faster than their wild-type counterparts. Increased lactate production and utilization of glucose as a fuel source indicated that Myog-deleted animals exhibited an increased glycolytic flux. Hypoglycemic Myog-deleted mice no longer possessed the ability to outrun their wild-type counterparts, implying the ability of these animals to further deplete their glucose reserves confers their enhanced exercise capacity. Moreover, Myog-deleted mice exhibited an enhanced response to long-term exercise training. The mice developed a greater proportion of type 1 oxidative muscle fibers, and displayed increased levels of succinate dehydrogenase activity, indicative of increased oxidative metabolism. Mdx:Myog-deleted mice exhibited a similar phenotype, outperforming their mdx counterparts, although lagging behind wild-type animals. The morphology of muscle tissue from mdx:Myog-deleted mice appears to mimic that of mdx animals, indicating that myogenin is dispensable for adult skeletal muscle regeneration. Through global gene expression profiling and quantitative (q)RT-PCR, I identified a unique set of putative myogenin-dependent genes involved in regulating metabolic processes. These data suggest myogenin’s functions during adulthood are distinctly different than those during embryogenesis, and myogenin acts as a high-level transcription factor regulating metabolic activity in adult skeletal muscle.

Studies on the muscle-specific regulatory protein myogenin

Recently, a family of muscle-specific regulatory factors that includes myogenin, myoD, myf-5, and MRF-4 has been identified. They share a high degree of homology within a region that contains a basic and helix-loop-helix domain. Transfection of many non-muscle cell types with any one of these genes results in the activation of the entire myogenic program. To explore the mechanism through which myogenin regulates myogenesis, we have prepared antibodies against peptides specific to myogenin. Using these antibodies we show that myogenin is a 32 Kd phospho-protein which is localized to the nuclei of muscle cells. In vitro, myogenin oligomerizes with the ubiquitous enhancer binding factor E12, and acquires high affinity for an element of the core of the muscle creatine kinase (MCK) enhancer that is conserved among many muscle-specific genes. Myogenin synthesized in BC$\sb3$H1 and C2 muscle cell lines also binds to the same site in the enhancer. However, the MCK enhancer is not activated in 10T1/2 fibroblasts which have been transfected with a constitutive myogenin expression vector until growth factors have been removed from the media. This result indicates that mitogenic signals block the actions of myogenin.. Mutagenesis of the myogenin/E12 binding site in the MCK enhancer abolishes binding of the hetero-oligomer and prevents trans-activation of the enhancer by myogenin. By site directed mutagenesis of myogenin we have shown that the basic region consists of three clusters of basic residues, two of which are required for binding and activation of the myogenic program. Myogenic activation, but not DNA binding, is lost when the 10 residue region between the two required basic clusters is substituted with the corresponding region from E12, which also contains a similar basic and helix-loop-helix domain. Functional revertants of this substitution mutant have identified two amino acids which confer muscle specificity. The properties of myogenin suggest that it functions as a sequence-specific DNA binding factor that interacts directly with muscle-specific genes during myogenesis and contains within its basic domain a region which imparts myogenic activation and is separable from DNA binding. ^

Regulation of {\it myogenin\/} transcription during mouse embryogenesis

The formation of skeletal muscle during vertebrate development involves the induction of mesoderm and subsequent generation of myoblasts that ultimately differentiate into mature muscles. The recent identification of a group of myogenic regulators that can convert fibroblasts to myoblasts has contributed to our understanding of the molecular events that underlie the establishment of the skeletal muscle phenotype. Members of this group of myogenic regulators share a helix-loop-helix (HLH) motif that mediates DNA binding. The myogenic HLH proteins bind to the consensus sequence CANNTG, referred to as an E-box, and activate muscle-specific transcription. In addition to E-boxes, other motifs, such as the MEF-2 binding site, have been shown to mediate muscle-specific transcription. The myogenic HLH proteins are expressed in the myogenic precursors in somites and limb buds, and in differentiated muscle fibers during embryogenesis, consistent with their roles as regulators for muscle development. The myogenic HLH proteins appear to auto-activate their own and cross-activate one another's expression in cultured cells. Myogenin is one of the myogenic HLH proteins and likely the regulator for terminal muscle differentiation. Myogenin is a common target of diverse regulatory pathways. To search for upstream regulators of myogenin, we studied regulation of myogenin transcription during mouse embryogenesis. We showed that the myogenin promoter contains a binding site for MEF-2, which can mediate indirectly the autoregulation of myogenin transcription. We found that a transgene under the control of a 1.5 kb 5$\sp\prime$ flanking sequence can recapitulate the temporal and spatial expression pattern of the endogenous myogenin gene during mouse embryogenesis. By tracing embryonic cells that activate myogenin-lacZ during embryogenesis, we found no evidence that lacZ was expressed in myogenic precursors migrating from somites to limb buds, suggesting the existence of regulators other than myogenic HLH proteins that can maintain cells in the myogenic lineage. Mutations of an E-box and a MEF-2 site in the myogenin promoter suppressed transcription in subsets of myogenic precursors in mouse embryos. These results suggest that myogenic HLH proteins and MEF-2 participate in separable regulatory pathways controlling myogenin transcription and provide evidence for positional regulation of myogenic regulators in the embryo. ^

The cloning and expression of myogenin, a gene regulating myogenesis

Expression of the differentiated skeletal muscle phenotype is a process that appears to occur in at least two stages. First, pluripotent stem cells become committed to the myogenic lineage. Although undifferentiated and capable of continued proliferation, determined myoblasts are restricted to a single developmental fate. Upon receiving the appropriate environmental signals, these determined myoblasts withdraw from the cell cycle, fuse to form multi-nucleated myotubes, and begin to express a battery of muscle-specific gene products that make up the functional and contractile apparatus of the muscle. This project is aimed at the identification and characterization of factors that control the determination and differentiation of myogenic cells. We have cloned a cDNA, called myogenin, that plays an important role in these processes. Myogenin is expressed exclusively in skeletal muscle in vivo and myogenic cell lines in vitro. Its expression is sharply upregulated during differentiation. When constitutively expressed in fibroblasts, myogenin converts these cells to the myogenic lineage. Transfected cells behave as myogenic tissue culture cells with respect to the genes they express, the way they respond to environmental cues, and are capable of fusing to form multinucleated myotubes. Sequence analysis showed that this cDNA has homology to a family of transcription factors in a region of 72 amino acids known as the basic helix-loop-helix motif. This domain appears to mediate binding to a DNA sequence element known as an E-box (CANNTG) essential for the activity of the enhancers of many muscle-specific genes.^ Analysis of myogenin in tissue culture cells showed that its expression is responsive to many of the environmental cues, such as the presence of growth factors and oncogenes, that modulate myogenesis. In an attempt to identify the cis- and trans-elements that control myogenin expression and thereby understand what factors are responsible for the establishment of the myogenic lineage, we have cloned the myogenin gene. After analysis of the gene structure, we constructed a series of reporter constructs from the 5$\prime$ upstream sequence of the myogenin gene to determine which cis-acting sequences might be important in myogenin regulation. We found that 184 nucleotides of the 5$\prime$ sequence was sufficient to direct high-level muscle-specific expression of the reporter gene. Two sequence elements present in the 184 fragment, an E-box and a MEF-2 site, have been shown previously to be important in muscle-specific transcription. Mutagenesis of these sites revealed that both sites are necessary for full activity of the myogenin promoter, and suggests that a complex hierarchy of transcription factors control myogenic differentiation. ^

A study of the {\it in vivo\/} role of myogenin

Myogenin is a member of the MyoD family of skeletal muscle specific bHLH transcription factors. All of the members of this family have been shown to initiate the muscle differentiation cascade in a variety of nonmuscle cell lines. Many of the properties of the MyoD family have been studied in vitro, but their in vivo roles had not yet been examined. In this thesis, I study the in vivo role of myogenin by creating mice that carry a mutation at the myogenin locus.^ Mice lacking the myogenin protein are born alive, but immobile. Histological examination showed that these mice are severely deficient in skeletal muscle; they show a reduction in the number and density of myofibers. In addition to the reduction in fiber number, these mice express lower levels of a variety of muscle-specific markers. The undifferentiated cells in the muscle forming regions of these mice do express some muscle-specific markers, indicating that these cells are determined but undifferentiated myoblasts. Additional studies show that the major muscle defect arises late in embryogenesis, at a time coincident with secondary myogenesis. Moreover, studies regarding the nature of the remaining myofibers indicate that they are representative of a normal population of myofibers, merely reduced in numbers. In addition, I studied the effects of combining the myogenin mutation with mutations in two other members of the MyoD family, MyoD and myf5. Mice mutant in myogenin + MyoD and myogenin + myf5 show no increase in the severity of the myogenin single mutation, as indicated by histological or molecular examination. These results reveal the unique and essential role of myogenin in mammalian skeletal myogenesis. ^

myogenin: Human candidate gene and knockout mouse effect

The myogenin gene encodes an evolutionarily conserved basic helix-loop-helix transcription factor that regulates the expression of skeletal muscle-specific genes and its homozygous deletion results in mice who die of respiratory failure at birth. The histology of skeletal muscle in the myogenin null mice is reminiscent of that found in some severe congenital myopathy patients, many of whom also die of respiratory complications and provides the rationale that an aberrant human myogenin (myf4) coding region could be associated with some congenital myopathy conditions.^ With PCR, we found similarly sized amplimers for the three exons of the myogenin gene in 37 patient and 40 control samples. In contrast to the GeneBank sequence for human myogenin, we report several differences in flanking and coding regions plus an additional 659 and 498 bps in the first and second introns, respectively, in all patients and controls. We also find a novel (CA)-dinucleotide repeat in the second intron. No causative mutations were detected in the myogenin coding regions of genomic DNA from patients with severe congenital myopathy.^ Severe congenital myopathies in humans are often associated with respiratory complications and pulmonary hypoplasia. We have employed the myogenin null mouse, which lacks normal development of skeletal muscle fibers as a genetically defined severe congenital myopathy mouse model to evaluate the effect of absent fetal breathing movement on pulmonary development.^ Significant differences are observed at embryonic days E14, E17 and E20 of lung:body weight, total DNA and histologically, suggesting that the myogenin null lungs are hypoplastic. RT-PCR, in-situ immunofluorescence and EM reveal pneumocyte type II differentiation in both null and wild lungs as early as E14. However, at E14, myogenin null lungs have decreased BrdU incorporation while E17 through term, augmented cell death is detected in the myogenin null lungs, not seen in wild littermates. Absent mechanical forces appear to impair normal growth, but not maturation, of the developing lungs in myogenin null mouse.^ These investigations provide the basis for delineating the DNA sequence of the myogenin gene and and highlight the importance of skeletal muscle development in utero for normal lung organogenesis. My observation of no mutations within the coding regions of the human myogenin gene in DNA from patients with severe congenital myopathy do not support any association with this condition. ^

Use of a hypomorphic allele of myogenin to analyze Myogenin-dependent processes in mouse development

Myogenin is a muscle-specific transcription factor essential for skeletal muscle differentiation. A severe reduction in the number of fused myotubes is seen in myogenin-null mice, and the expression of genes characteristic of differentiated skeletal muscle is reduced. Additionally, sternebrae defects are seen in myogenin-null mice, a secondary defect in the sternal cartilage precursors. Very little is known about the quantitative requirement for myogenin in muscle differentiation and thoracic skeletal development in vivo. In this thesis I describe experiments utilizing a mouse line harboring a hypomorphic allele of myogenin, generated by gene targeting techniques in embryonic stem cells. The nature of the hypomorphism was due to lowered levels of myogenin from this allele. In embryos homozygous for the hypomorphic allele, normal sternum formation and extensive muscle differentiation was observed. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable after birth. These results suggest skeletal muscle differentiation is highly sensitive to the absolute amounts of myogenin, and reveal distinct threshold requirements for myogenin in skeletal muscle differentiation, sternum formation, and viability in vivo. The hypomorphic allele was utilized as a genetically sensitized background to identify other components of myogenin-mediated processes. Using a candidate gene approach I crossed null mutations in MEF2C and MRF4 into the hypomorphic background and examined whether these mutations affected muscle differentiation and skeleton formation in the myogenin hypomorph. Although MEF2C mutation did not affect any phenotypes seen in the hypomorphic background, MRF4 was observed to be an essential component of myogenin-mediated processes of thoracic skeletal development. Additionally, the hypomorphic allele was very sensitive to genetic effects, suggesting the existence of mappable genetic modifiers of the hypomorphic allele of myogenin. ^

Analyzing the function of myogenin in adult satellite cells through the construction of a myogenin conditional allele

Although mechanisms regulating the formation of embryonic skeletal muscle are well characterized, less is known about muscle formation in postnatal life. This disparity is unfortunate because the largest increases in skeletal muscle mass occur after birth. Adult muscle stem cells (satellite cells) appear to recapitulate the events that occur in embryonic myoblasts. In particular, the myogenic basic helix-loop-helix factors, which have crucial functions in embryonic muscle development, are assumed to have similar roles in postnatal muscle formation. Here, I test this assumption by determining the role of the myogenic regulator myogenin in postnatal life. Myogenin-null mice die at birth, necessitating the generation of floxed alleles of myogenin and the use of cre-recombinase lines to delete myogenin. Removing myogenin before embryonic muscle development resulted in myofiber deficiencies identical to those observed in myogenin-null mice. However, mice in which myogenin was deleted following embryonic muscle development had normal skeletal muscle, except for modest alterations in MRF4 and MyoD expression. Notably, myogenin-deleted mice were 30% smaller than controls, suggesting that myogenin's absence disrupted general body growth. These results suggest that skeletal muscle growth in postnatal life is controlled by mechanisms distinct from those occurring in embryonic muscle development. ^

Regulation of XMyoDa transcription by the binding of XMEF2A to the TATA box

MEF2 is a $\underline{\rm m}$yocyte-specific $\underline{\rm e}$nhancer-binding $\underline{\rm f}$actor that binds a conserved DNA sequence, CTA(A/T)$\sb4$TAG. A MEF2 binding site in the XMyoDa promoter overlaps with the TATA box and is required for muscle specific expression. To examine the potential role of MEF2 in the regulation of MyoD transcription during early development, the appearance of MEF2 binding activity in developing Xenopus embryos was analyzed with the electrophoretic mobility shift assay. Two genes were isolated from a X. Laevis stage 24 cDNA library that encode factors that bind the XMyoDa TFIID/MEF2 site. Both genes are highly homologous to each other, belong to the MADS ($\underline{\rm M}$CM1-$\underline{\rm A}$rg80-agamous-$\underline{\rm d}$eficiens-$\underline{\rm S}$RF) protein family, and most highly related to the mammalian MEF2A gene, hence they are designated as XMEF2A1 and XMEF2A2. Proteins encoded by both cDNAs form specific complexes with the MEF2 binding site and show the same binding specificity as the endogenous MEF2 binding activity. XMEF2A transcripts accumulate preferentially in developing somites after the appearance of XMyoD transcripts. XMEF2 protein begins to accumulate in somites at tailbud stages. Transcriptional activation of XMyoD promoter by XMEF2A required only the MADS box and MEF2-specific domain when XMEF2A is bound at the TATA box. However, a different downstream transactivation domain was required when XMEF2A activates transcription through binding to multiple upstream sites. These results suggest that different activation mechanisms are involved, depending on where the factor is bound. Mutations in several basic amino acid clusters in the MADS box inhibit DNA binding suggesting these amino acids are essential for DNA binding. Mutation of Thr-20 and Ser-36 to the negatively charged amino acid residue, aspartic acid, abolish DNA binding. XMEF2A activity may be regulated by phosphorylation of these amino acids. A dominant negative mutant was made by mutating one of the basic amino acid clusters and deleting the downstream transactivation domain. In vivo roles of MEF2 in the regulation of MyoD transcription were investigated by overexpression of wild type MEF2 and dominant negative mutant of XMEF2A in animal caps and assaying for the effects on the level of expression of MyoD genes. Overexpression of MEF2 activates the transcription of endogenous MyoD gene family while expression of a dominant negative mutant reduces the level of transcription of XMRF4 and myogenin genes. These results suggest that MEF2 is downstream of MyoD and Myf5 and that MEF2 is involved in maintaining and amplifying expression of MyoD and Myf5. MEF2 is upstream of MRF4 and myogenin and plays a role in activating their expression. ^

Phosphorylation in the regulation of muscle-specific transcription

The contents of this dissertation include studies on the mechanisms by which FGF and growth factor down-stream kinases inactivate myogenin; characterization of myogenin phosphorylation and its role in regulation of myogenin activity; analysis the C-terminal transcriptional activation domain of myogenin; studies on the nuclear localization of myogenin and characterization of proteins that interact with PKC.^ Activation of muscle transcription by the MyoD family requires their heterodimerization with ubiquitous bHLH proteins such as the E2A gene products E12 and E47. I have shown that dimerization with E2A products potentiates phosphorylation of myogenin at serine 43 in its amino-terminus and serine 170 in the carboxyl-terminal transcription activation domains. Mutations of these sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. Consistent with the role of phosphorylation at serine 170, analysis of the carboxyl-terminal transcriptional activation domain by deletion has revealed a stretch of residues from 157 to 170 which functions as a negative element for myogenin activity.^ In addition to inducing phosphorylation of myogenin, E12 also localizes myogenin to the nucleus. The DNA binding and dimerization mutants of myogenin show various deficiencies in nuclear localization. Cotransfection of E12 with the DNA binding mutants, but not a dimerization mutant, greatly enhances their nuclear binding. These data suggest that the nuclear localization signal is located in the DNA binding region and myogenin can also be nuclear localized by virtue of dimerizing with a nuclear protein.^ FGF is one of the most potent inhibitors of myogenesis and activates many down-stream pathways to exert its functions. One of these pathway is the MAP kinase pathway. Studies have shown that Raf-1 and Erk-1 kinase inactivate transactivation by myogenin and E proteins independent of DNA binding. The other is the PKC pathway. In transfected cells, FGF induces phosphorylation of thr-87 that maps to the previously identified PKC sites in the DNA binding domain of myogenin. Myogenin mutant T-N87 could resist the inhibition directed to the bHLH domain by FGF, suggesting that FGF inactivates myogenin by inducing phosphorylation of this site. In C2 myotubes, where FGF receptors are lost, the phosphatase inhibitor, okadaic acid, and phorbal ester PdBu, can also induce the phosphorylation of thr-87. This result supports the previous observation and suggests that in myotubes, other mechanisms, such as innervation, may inactivate myogenin through PKC induced phosphorylation.^ Many functions of PKC have been well documented, yet, little is known about the activators or effectors of PKC or proteins that mediate PKC nuclear localizations. Identification of PKC binding proteins will help to understand the molecular mechanism of PKC function. Two proteins that interact with the C kinase (PICKS) have been characterized, PICK-1 and PICK-2. PICK1 interacts with two conserved regions in the catalytic domain of PKC. It is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK2 is a novel protein with homology to the heat shock protein family. It interacts extensively with the catalytic domain of PKC and is localized in the cytoplasm in a punctate pattern. PICK1 and PICK2 may play important roles in mediating the actions of PKC. ^

Functional analysis of MRF4 during mouse embryogenesis

MRF4 is one of four skeletal muscle specific regulatory genes, (the other three genes being MyoD, myf5, and myogenin), each of which has the unique ability to orchestrate an entire program of muscle-specific transcription when introduced into diverse cell types. These findings have led to the notion that these factors function as master regulators of muscle cell fate. Analysis of mice lacking MyoD, myf5, and myogenin have further defined their roles in the commitment and differentiation of myotomal progenitor cells. Current data strongly supports the model that MyoD and myf5 share functional redundancy in determining the muscle cell lineage, while myogenin acts downstream of MyoD and myf5, to initiate myoblast differentiation. Unlike other myogenic bHLH genes, MRF4 is expressed predominantly in the adult, suggesting that it may function to regulate adult muscle maturation and maintenance. To test this hypothesis and to eventually incorporate MRF4 into a general model for muscle specification, differentiation, maturation and maintenance, I deleted the MRF4 gene. MRF4-null mice are viable and fertile, however, they show mild rib anomalies. In addition, the expression of myogenin is dramatically upregulated only in the adult, suggesting that myogenin may compensate for the loss of MRF4 in the adult, and MRF4 may normally suppress the expression of myogenin after birth. MRF4 is also required during muscle regeneration after injury.^ To determine the degree of genetic redundancy between MRF4-myogenin; and MRF4-MyoD, I crossed the MRF4-null mice with MyoD- and myogenin-null mice respectively. There are no additional muscle phenotypes in double-null progeny from a MRF4 and myogenin cross, suggesting that the existence of residual fibers in myogenin-null mice is not due to the presence of MRF4. MRF4 expression also cannot account for the ability of myogenin-null myoblasts to differentiate in vitro. However, the combination of the MRF4-null mutation with the myogenin-null mutation results in a novel rib phenotype. This result suggests that MRF4 modifies the myogenin-null rib phenotype, and MRF4 and myogenin play redundant roles in rib development.^ MRF4 also shares dosage effects with MyoD during mouse development. (MyoD+/$-$;MRF4$-$/$-$)mice are fertile and viable, while (MyoD$-$/$-$;MRF4+/$-$) mice die between birth and two weeks after birth, and have a small skeletal structure. The double homozygous mice for MRF4 and MyoD mutations are embryonic lethal and die at around E10.5. These results suggest that MRF4 and MyoD share overlapping functions during mouse embryogenesis. ^

Expression of neuromuscular junction messenger RNAs and protein in aged rats

The loss of skeletal muscle mass is believed to be the dominant reason for reduced strength in aging humans. The purpose of this investigation was to gain some information as to why skeletal muscles lose mass as we age. Since nervous system innervation is essential for skeletal muscle fiber viability, incomplete regional reinnervation during normal synaptic junction turnover has been hypothesized to result in selective muscle fiber loss. Examined here was the age-related association in skeletal muscle between atrophy and the expression of mRNAs encoding the γ- and ϵ-subunits of the nicotinic acetylcholine receptor, myogenin, and muscle specific receptor kinase (MuSK). Gastrocnemius and biceps brachii muscles were collected from young (2 month), adult (18 month), and old (31 month) Fischer 344 cross brown Norway F 1 male rats. In the gastrocnemius, muscles of old vs. young and adult rats, lower muscle mass was accompanied by significantly elevated acetylcholine receptor γ-subunit, myogenin, and MuSK mRNA levels. In contrast, the biceps brachii muscle in the same animals exhibited neither atrophy nor a change in acetylcholine receptor γ-subunit, myogenin, or MuSK mRNA levels. Expression of the acetylcholine receptor ϵ-subunit mRNA did not change with age in either gastrocnemius or biceps brachii muscles. Since acetylcholine receptor γ-subunit, myogenin, and MuSK mRNA levels are upregulated in surgically denervated skeletal muscles of young rats while expression of the acetylcholine receptor ϵ-subunit does not change, the findings of the current investigation suggest that a select fiber population within atrophied skeletal muscles of old rats may be in a denervated-like state. I speculate that increases in γ-subunit, myogenin, and MuSK mRNA levels in atrophied muscles of old rats are compensatory responses to nerve terminal retraction. Indeed, a prolongation of denervation in these muscle fibers would subsequently result in their atrophy and death, ultimately leading to a decline in the number of force generating elements present in the muscle. ^

Regulation of myogenesis by growth signals -growth factors, oncogenes and protein kinases

Establishment of a myogenic phenotype involves antagonism between cell proliferation and differentiation. The recent identification of the MyoD family of muscle-specific transcription factors provides opportunities to dissect at the molecular level the mechanisms through which defined cell type-specific transcription factors respond to environmental cues and regulate differentiation programs. This project is aimed at elucidation of the molecular mechanism whereby growth factors repress myogenesis. Initial studies demonstrated that nuclear oncogenes such as c-fos, junB and c-jun are immediate early genes that respond to serum and TGF-$\beta$. Using the muscle creatine kinase (MCK) enhancer linked to the reporter gene CAT as a marker for differentiation, we showed that transcriptional function of myogenin can be disrupted in the presence of c-Fos, JunB and cjun. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, failed to show the inhibition. The repression by Fos and Jun is targeted at KE-2 motif, the same sequence that mediates myogenin-dependent activation and muscle-specific transactivation. Deletion analysis indicated that the transactivation domain of c-Jun at the N-terminus is responsible for the repression. Considering that myogenin is a phosphoprotein and cAMP and TPA are able to regulate myogenesis, we examined whether constitutively active protein kinase C (PKC) and protein kinase A (PKA) could substitute for exogenous growth factors and prevent transcription activation by myogenin. Indeed, the basic region of myogenin is phosphorylated by PKC at a threonine that is conserved in all members of the MyoD family. Phosphorylation at this site attenuates DNA binding activity of myogenin. Protein kinase A can also phosphorylate myogenin in a region adjacent to the DNA binding domain. However, phosphorylation at this site is insufficient to abrogate myogenin's DNA binding capacity, suggesting that PKA and PKC may affect myogenin transcriptional activity through different mechanisms. These findings provide insight into the mechanisms through which growth factor signals negatively regulate the muscle differentiation program and contribute to an understanding of signal transducing pathways between the cell membrane and nucleus. ^