Muscle Creatine Kinase Gene Research Articles

Constitutive activation of estrogen receptor α signaling in muscle prolongs exercise endurance in mice

Estrogen is a female hormone that plays a role in various tissues, although the mechanism in skeletal muscle has not been fully clarified. We previously showed that systemic administration of estrogen for 10 weeks ameliorated decreased exercise endurance in ovariectomized mice. To assess whether a long-term and muscle-specific activation of estrogen signaling modulates muscle function, we constructed an expression plasmid for a constitutively active estrogen receptor α (caERα) under the control of muscle creatine kinase (Mck) gene promoter/enhancer. In C2C12 mouse myoblastic cells, transfection of the Mck-caERα plasmid elevated the estrogen response element-driven transcription in a ligand-independent manner. Using this construct, we generated Mck-caERα transgenic mice, in which caERα is predominantly expressed in muscle. Treadmill running test revealed that female Mck-caERα mice exhibit a prolonged running time and distance compared with the wild-type mice. Moreover, microarray expression analysis revealed that the genes related to lipid metabolism, insulin signaling, and growth factor signaling were particularly upregulated in the quadriceps femoris muscle of Mck-caERα mice. These results suggest that estrogen signaling potentiates exercise endurance in skeletal muscle through modulating the expression of metabolism-associated genes.

Association of lamin A/C with muscle gene-specific promoters in myoblasts

The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome–lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin–chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina–chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.

Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development.

Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear. Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co-activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification before myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co-immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP. MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo.

G.P.64 Histological, immunohistochemical and electron microscopic study of muscle in MCK-βAPP mice

Abstract The MCK-βAPP transgenic mouse, which carries the human amyloid precursor protein (APP) cDNA transgene, has been proposed as a model for sporadic inclusion body myositis (s-IBM). βAPP expression is confined to the skeletal muscles by the flanking muscle creatine kinase (MCK) gene, and provides a suitable model for investigating the downstream effects of selective over-expression of βAPP in muscle. We investigated the histological, immunohistochemical and ultrastructural changes in skeletal muscles in transgenic MCK-βAPP (tg; n = 24) and wild-type mice (wt; n = 22) aged 3–18 months. The only consistent change, which was observed equally in an age-dependent manner in both tg and wt male mice, was the presence of tubular aggregates in type II muscle fibres that has also been reported in other mouse strains. In contrast to a previous study of the MCK-βAPP mouse, no s-IBM related changes, such as inflammation, necrosis/regeneration, rimmed vacuoles or amyloid inclusions were observed and MHC-I antigen expression was not up-regulated, suggesting that these changes are not related to over-expression of βAPP. Although full length βAPP (fl-APP) has been confirmed on western blots, our failure to detect amyloid inclusions suggests that fl-APP may not be processed into β-amyloid, or that instead of accumulating in muscle fibres, β-amyloid may be secreted into the circulatory system. Our findings indicate that the MCK-βAPP mouse may not be an appropriate model of human s-IBM.

Single nucleotide polymorphisms in the myostatin (MSTN) and muscle creatine kinase (CKM) genes are not associated with elite endurance performance

Maximal oxygen uptake (VO2max) is one of the most important determinants of elite endurance performance. VO2max is determined by a whole range of genetic and environmental factors. Single nucleotide polymorphisms (SNPs) in muscle myostatin (MSTN) and creatine kinase (CKM) genes are candidates for VO2max and skeletal muscle performance phenotypes. Common MSTN (rs3791783, rs11681628 and rs7570532) and CKM (rs344816, rs10410448, rs432979, rs1133190, rs7260359, rs7260463 and rs4884) SNPs, selected from HapMap CEU data in order to tag the genetic variability of the proteins, were genotyped in 316 male Caucasian elite endurance athletes and 304 sedentary controls from the Genathlete study. Association with elite endurance performance was determined by logistic regression analysis. The P-value for statistical significance was set at <0.01. None of the SNPs or haplotypes showed a significant association with elite endurance status. We conclude that common variants of MSTN and CKM genes do not play a role in attaining high-level endurance performance in Caucasian populations.

Hesperedin promotes MyoD‐induced myogenic differentiation in vitro and in vivo

The bioflavonoid, hesperedin, promotes osteoblast differentiation in human mesenchymal stem cells, indicating an anabolic effect of hesperedin on bone metabolism. Murine bone marrow mesenchymal stem cells undergo myogenic differentiation as well as osteogenic differentiation. We therefore explored whether hesperedin modulates muscle cell differentiation. Myoblast C2C12 cells were differentiated into muscle cells in the presence or absence of hesperedin. The effects of hesperedin on myogenic differentiation were determined by analysing specific muscle markers in vitro using reporter gene assays, immunoblotting, RT-PCR and DNA pull-down assays. In vivo, the effects of hesperedin were assessed using the freeze injury-induced muscle regeneration model in mice and daily injections of hesperedin for 6 days. Hesperedin promoted myogenic differentiation, in a dose-dependent manner, by increasing myogenin gene expression. MyoD-induced myogenin gene transcription was enhanced by hesperedin, as this bioflavonoid augmented the nuclear localization and myogenin promoter-binding of MyoD. In addition, hesperedin increased myogenin and muscle creatine kinase gene expression during myogenic differentiation from C3H10T1/2 mesenchymal stem cells in a MyoD-dependent manner and accelerated in vivo muscle regeneration induced by muscle injury. Our results demonstrate that hesperedin promoted myogenic differentiation in vitro and in vivo through activation of MyoD-mediated myogenin expression, suggesting a beneficial role in promoting muscle regeneration, following injury.

An Adeno-Associated Virus Vector Efficiently and Specifically Transduces Mouse Skeletal Muscle

Expression of a therapeutic gene in the skeletal muscle is a practical strategy to compensate a patients' insufficient circulating factor. Its clinical application requires a muscle-targeting vector capable of inducing a continuous high-level transgene expression. We modified an adeno-associated virus serotype 2 (AAV2) vector expressing luciferase from the mouse muscle creatine kinase gene promoter-enhancer (Ckm). First, AAVS1 insulator was inserted into the vector genome for transcriptional enhancement. This increased transduction of mouse quadriceps muscle by 11-fold at 4weeks after intramuscular injection. Second, two capsid modifications were combined (21F capsid): incorporation of a segment of AAV1 capsid to produce a hybrid capsid and substitution of a tyrosine with a phenylalanine. Use of 21F capsid increased muscle transduction further by 18-fold, resulting in 200-fold higher efficacy than that of the unmodified vector. Compared with a vector having human elongation factor 1α promoter which showed similar efficacy in the muscle, this vector having Ckm transduced non-muscle organs less efficiently after intravenous administration. The AAV2 vector composed of the modified genome and capsid provides a backbone to develop a clinical vector expressing a therapeutic gene in the muscle.

Down-regulation of MyoD by Calpain 3 Promotes Generation of Reserve Cells in C2C12 Myoblasts

Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

P53 and Metabolism: The GAMT Connection

In this issue of Molecular Cell, Ide et al. (2009) have identified the enzyme guanidinoacetate methyltransferase (GAMT) that regulates creatine metabolism as a p53 target involved in apoptosis, reactive oxygen species (ROS), and fatty acid metabolism.

Genetic Complementation of Human Muscle Cells via Directed Stem Cell Fusion

Duchenne muscular dystrophy (DMD) is caused by mutations in the X chromosome-linked DMD gene, which encodes the sarcolemma-stabilizing protein-dystrophin. Initial attempts at DMD therapy deployed muscle progenitor cells from healthy donors. The utilization of these cells is, however, hampered by their immunogenicity, while those from DMD patients are scarce and display limited ex vivo replication. Nonmuscle cells with myogenic capacity may offer valuable alternatives especially if, to allow autologous transplantation, they are amenable to genetic intervention. As a paradigm for therapeutic gene transfer by heterotypic cell fusion we are investigating whether human mesenchymal stem cells (hMSCs) can serve as donors of recombinant DMD genes for recipient human muscle cells. Here, we show that forced MyoD expression in hMSCs greatly increases their tendency to participate in human myotube formation turning them into improved DNA delivery vehicles. Efficient loading of hMSCs with recombinant DMD was achieved through a new tropism-modified high-capacity adenoviral (hcAd) vector directing striated muscle-specific synthesis of full-length dystrophin. This study introduces the principle of genetic complementation of gene-defective cells via directed cell fusion and provides an initial framework to test whether transient MyoD synthesis in autologous, gene-corrected hMSCs increases their potential for treating DMD and, possibly, other muscular dystrophies.

Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle

Mucopolysaccharidosis VI (MPS VI) is caused by deficient activity of arylsulfatase B (ARSB), resulting in intralysosomal storage of dermatan sulfate (DS) and multisystem disease without central nervous system involvement. After gene transfer, muscle or liver can theoretically be converted into factories for systemic ARSB secretion, leading to uptake by non-transduced cells. We have injected newborn MPS VI rats and cats with adeno-associated viral (AAV) vectors expressing ARSB under the control of liver-specific, muscle-specific, or universally active promoters. After systemic or intramuscular (IM) administration of AAV, therapeutic levels of circulating ARSB are achieved, resulting in skeletal improvements and significant decrease in glycosaminoglycan (GAG) storage, inflammation and apoptosis (despite a neutralizing immune response to ARSB in MPS VI rats). In addition, we have observed wide-spread dissemination of vector after IM AAV administration. This results in secretion of therapeutic levels of ARSB when the universally active cytomegalovirus (CMV) but not the muscle-specific muscle creatine kinase (MCK) promoter is used, suggesting that transduction of extramuscular sites rather than enzyme secretion from muscle occurs after muscle ARSB gene transfer. We conclude that AAV-mediated expression of ARSB from liver represents a feasible therapeutic strategy for MPS VI, potentially avoiding multiple infusions of costly recombinant enzyme associated with enzyme replacement therapy.

Long-term Skeletal Muscle Protection After Gene Transfer in a Mouse Model of LGMD-2D

Limb girdle muscular dystrophy (LGMD) describes a group of inherited diseases resulting from mutations in genes encoding proteins involved in maintaining skeletal muscle membrane stability. LGMD type-2D is caused by mutations in alpha-sarcoglycan (sgca). Here we describe muscle-specific gene delivery of the human sgca gene into dystrophic muscle using an adeno-associated virus 1 (AAV1) capsid and creatine kinase promoter. Delivery of this construct to adult sgca(-/-) mice resulted in localization of the sarcoglycan complex to the sarcolemma and a reduction in muscle fiber damage. Sgca expression prevented disease progression as observed in vivo by T(2)-weighted magnetic resonance imaging (MRI) and confirmed in vitro by decreased Evan's blue dye accumulation. The ability of recombinant AAV-mediated gene delivery to restore normal muscle mechanical properties in sgca(-/-) mice was verified by in vitro force mechanics on isolated extensor digitorum longus (EDL) muscles, with a decrease in passive resistance to stretch as compared with untreated controls. In summary, AAV/AAV-sgca gene transfer provides long-term muscle protection from LGMD and can be non-invasively evaluated using magnetic resonance imaging.

Transcriptional repression by the basic helix-loop-helix protein Dec2: Multiple mechanisms through E-box elements

Dec2, a member of the basic helix-loop-helix (bHLH) superfamily, has been shown to function as a transcriptional repressor and is implicated in cell proliferation and differentiation. In addition, Dec2 transcripts exhibit a striking circadian oscillation in the suprachiasmatic nucleus. To identify the molecular mechanisms by which Dec2 regulates gene expression, we carried out structure-function analyses. Gel retardation and luciferase assays showed that Dec2, as well as its related protein Dec1, preferentially binds to class B E-box elements (CACGTG) as a homodimer and represses the transcription of target genes in a histone deacetylase (HDAC)-dependent manner. Functional studies with the GAL4-DNA binding domain fusion proteins identified the domain responsible for the repression activity of Dec2 in its C-terminal region, which is also necessary to recruit HDAC1. In addition, the basic and HLH domains of Dec2 were required for DNA binding and homodimerization, respectively. In contrast, Dec proteins repressed a MyoD-activated promoter activity of muscle creatine kinase gene through class A E-box in an HDAC1-independent manner. Dec2 formed a heterodimer with MyoD through the basic and HLH domains. Consistent with this, both the basic and HLH domains were required for the ability of Dec2 to inhibit the transcriptional activity of MyoD. These findings indicate that Dec2 employs multiple mechanisms, including DNA-binding and protein-protein interactions, to achieve E-box-dependent transcriptional repressions.

561. Novel Tissue-Specific Regulatory Cassettes Direct High-Level Transgene Expression in Skeletal and Cardiac Muscle

Top of pageAbstract Systemic delivery of rAAV6 vectors can achieve efficient transduction of the entire striated musculature, making this an attractive strategy for gene therapy of Duchenne Muscular Dystrophy. However, this delivery method also transduces cells in many non-muscle tissues, which may cause therapeutic problems including toxicity and a transgene-directed immune response. These problems could be avoided by limiting transgene expression through the use of muscle-specific promoters. Unfortunately, the expression levels of such promoters following systemic delivery of rAAV vectors are lower than viral promoters in most skeletal muscles and negligible in cardiac muscle. The goal of this project is to design regulatory cassettes that drive high levels of skeletal and cardiac muscle-specific transgene expression while also being sufficiently short (<800bp) to fit into rAAV vectors containing microdystrophin cDNA (3.8-kb). Our lab has developed a series of muscle-specific regulatory cassettes based on the enhancer and promoter of the murine muscle creatine kinase (MCK) gene. The CK6 cassette, previously thought to be the strongest modification, has higher activity than the wild type cassette in skeletal muscle, but very low activity in cardiac and diaphragm muscle. We have designed new cassettes to increase expression, and their activities have been evaluated in various muscles and non-muscle tissues following systemic delivery of rAAV6 vectors expressing a human placental alkaline phosphatase reporter gene. First, we designed a 570-bp MCK-based cassette, CK7, which exhibited activity levels similar to CK6 in quadriceps, a predominantly fast-twitch muscle, but had significantly higher activity in cardiac muscle, diaphragm, and soleus, a predominantly slow-twitch muscle. To further improve expression, especially in cardiac muscle, we added a 190-bp enhancer from the murine alpha-myosin heavy chain gene (|[alpha]|MHC) to the CK7 cassette to produce MHCK7, a 770-bp cassette that has the highest overall activity. When compared to CK6, the activity of MHCK7 was about 400-fold higher in cardiac muscle and 10-fold higher in soleus, which was equivalent to the activity of the CMV promoter. Staining for AP activity in cross-sections of both muscles revealed strong expression in virtually all fibers. The activity in diaphragm was about 40-fold higher, but was still considerably lower than the CMV promoter, with only about 20% of the fibers staining positive. Finally, the activity in quadriceps muscle was equivalent to CK6. The activity of MHCK7 in kidney, aorta, brain, testes, and intestine was at background levels, while a very slight increase in activity, about 50|[ndash]|200-fold lower than in muscle, was detected in liver, spleen, and lung. Based on these results, the MHCK7 cassette is currently being tested with respect to its capacity for driving microdystrophin expression in mdx mice following intramuscular and systemic injections of rAAV6 vectors.

266. Development of rAAV-Based Skeletal and Cardiac Muscle Regulatory Cassettes for Gene Therapy of Duchenne Muscular Dystrophy

Top of pageAbstract A promising gene therapy approach for treatment of DMD involves systemic delivery of rAAV vectors encoding microdystrophin. This method was shown to mediate efficient transduction of all striated muscles and high-level transgene expression driven by the ubiquitous CMV promoter. However, the use of viral promoters increases the risk of an immune response, largely due to transgene expression in antigen-presenting cells, and may thus be inadequate for long-term therapy. An ideal regulatory cassette for gene therapy of DMD thus needs to direct high-level, tissue-specific expression in skeletal and cardiac muscle and be short enough to package into the rAAV microdystrophin construct. Our lab has developed a series of muscle-specific regulatory cassettes based on the murine muscle creatine kinase (MCK) gene enhancer and promoter regions. The previous |[ldquo]|best|[rdquo]| small cassette, CK6 (580 bp) drives microdystrophin expression in skeletal muscle following systemic rAAV delivery, at 10% of the level of the CMV promoter. However, expression in heart and diaphragm is very low. To improve expression, we combined the wild type MCK enhancer with a 190 bp enhancer region from the murine alpha myosin heavy chain (MyHC) gene, which is expressed at high levels in cardiac muscle. Addition of the MyHC enhancer increased activity in MM14 skeletal myocytes by 5-fold as assessed by alkaline phosphatase reporter gene activity. Furthermore, high-level expression was detected in skeletal muscle, as well as heart and diaphragm after systemic delivery in mice of rAAV6 vectors encoding these constructs. The activity of the hybrid cassette was 7-fold higher in heart and 3-fold higher in diaphragm and soleus (predominantly slow-twitch muscle fibers) when compared to the wild type MCK cassette, while the activity in tibialis anterioris (predominantly fast-twitch muscle fibers) was unchanged. Expression was restricted to muscle tissue, as evidenced by nearly undetectable levels in the liver, spleen, lung, and aorta. Additional cassettes comprising the MyHC enhancer and various modifications of the minimal MCK cassette have also been tested in MM14 skeletal myocytes. The strongest cassettes were shown to possess 12-fold higher activity than the wild type MCK cassette, and were about 55% as active as the CMV promoter. These contained a 63 bp deletion within the MCK enhancer, addition of a 50 bp region downstream of the transcription initiation site, and a mutation that creates a consensus initiator-binding site from the terminal transferase promoter. We are currently testing activity of these constructs in mouse muscle and non-muscle tissues following systemic delivery of rAAV6 vectors. The strongest tissue-specific cassette will be evaluated for long-term expression of therapeutic levels of microdystrophin in the mdx mouse model for DMD.

The mouse muscle creatine kinase promoter faithfully drives reporter gene expression in transgenicXenopus laevis

Developing Xenopus laevis experience two periods of muscle differentiation, once during embryogenesis and again at metamorphosis. During metamorphosis, thyroid hormone induces both muscle growth in the limbs and muscle death in the tail. In mammals, the muscle creatine kinase (MCK) gene is activated during the differentiation from myoblasts to myocytes and has served as both a marker for muscle development and to drive transgene expression in transgenic mice. Transcriptional control elements are generally highly conserved throughout evolution, potentially allowing mouse promoter use in transgenic X. laevis. This paper compares endogenous X. laevis MCK gene expression and the mouse MCK (mMCK) promoter driving a green fluorescent protein reporter in transgenic X. laevis. The mMCK promoter demonstrated strong skeletal muscle-specific transgene expression in both the juvenile tadpole and adult frog. Therefore, our results clearly demonstrate the functional conservation of regulatory sequences in vertebrate muscle gene promoters and illustrate the utility of using X. laevis transgenesis for detailed comparative study of mammalian promoter activity in vivo.

Expression of muscle creatine kinase gene of mice and interaction of nuclear proteins with MEF-2, E boxes and A/T-rich elements during aging.

The changes in the expression of muscle creatine kinase (MCK) gene in the heart and skeletal muscle of mice during aging were studied. Its expression declines as a function of age in the heart, however, no age-related change is observed in the skeletal muscle. The cis-acting elements, MEF-2, E boxes and A/T rich elements present in the enhancer region of the mouse MCK gene are known to regulate the expression of the gene. Hence, these elements were subcloned and electrophoretic mobility shift assay was carried out to investigate the changes in the binding of the nuclear trans-acting protein factors of the heart with these elements as a function of age. These factors showed specificity for the respective cis-acting elements. Furthermore, the binding of these factors was found to decrease during aging which may contribute to the age-related decline in the expression of the MCK gene and activity of the heart.

Reduction of diet-induced obesity in transgenic mice overexpressing uncoupling protein 3 in skeletal muscle.

It has been suggested that uncoupling protein 3 (UCP3) can increase energy expenditure, thereby regulating body weight. Although studies on UCP3 knock-out mice suggest that lack of UCP3 function does not cause obesity or Type 2 diabetes, it is possible that up-regulation of UCP3 function improves these disorders or their clinical sequelae. A 10- to 20-fold increase of UCP3 gene expression is achievable through physiological or pharmacological stimuli. We examined the phenotype of transgenic mice with approximately 18-fold overexpression of mouse UCP3 mRNA in skeletal muscle. We generated transgenic mice with approximately 18-fold overexpression of mouse UCP3 mRNA in skeletal muscle under control of the skeletal muscle-specific muscle creatine kinase gene promoter. The phenotype of these mice was analysed either on a standard diet or on a 4-week high-fat diet. In mice on standard chow, there was no difference in body weight, oxygen consumption and mitochondrial protonmotive force between transgenic mice and non-transgenic littermates. However, transgenic mice tended to have lower body weight, increased oxygen consumption and decreased mitochondrial protonmotive force than the control mice. Transgenic mice on a 4-week high-fat diet consumed much more oxygen and had noticeably less weight gain and less epididymal fat, as well as better glucose tolerance than non-transgenic littermates. Our study shows that 18-fold overexpression of UCP3 mRNA in the skeletal muscle reduced diet-induced obesity. An 18-fold increase of UCP3 mRNA can be attained by physiological or pharmacological stimuli, suggesting that UCP3 has therapeutic potential in the treatment of obesity.

Differences in the Function of Three Conserved E-boxes of the Muscle Creatine Kinase Gene in Cultured Myocytes and in Transgenic Mouse Skeletal and Cardiac Muscle

The 1256-base pair enhancer-promoter of the mouse muscle creatine kinase gene includes three CAnnTG E-boxes that are conserved among mammals and have flanking and middle sequences conforming to consensus muscle regulatory factor binding sites. This study seeks to determine whether these E-boxes are critical for muscle creatine kinase expression in physiologically distinct muscles. Mutations of the "right" and "left" E-boxes in the enhancer decreased expression in cultured skeletal myocytes approximately 10- and 2-fold, respectively, whereas a "promoter" E-box mutation had little effect. In neonatal myocardiocytes, the left E-box mutation decreased expression approximately 3-fold, whereas right or promoter E-box mutations had no effect. Very different effects were seen in transgenic mice, where the promoter E-box mutation decreased expression in quadriceps, extensor digitorum longus, and soleus approximately 10-fold, and approximately 100-fold in distal tongue, diaphragm, and ventricle. The right E-box mutation, tested in the presence of the other two mutations, caused a significant decrease in distal tongue, but not in quadriceps, extensor digitorum longus, soleus, or ventricle. Mutation of the left E-box actually raised expression in soleus, suggesting a possible repressor role for this control element. The discrepancies between mutation effects in differentiating skeletal muscle cultures, neonatal myocardiocytes, and adult mice suggested that the E-boxes might play different roles during muscle development and adult steady-state function. However, transgenic analysis of embryonic and early postnatal mice indicated no positive role for these three E-boxes in early development, implying that differences in E-box function between adult muscle and cultured cells are the result of physiological signals.

Basic Helix-Loop-Helix Transcription Factor Epicardin/Capsulin/Pod-1 Suppresses Differentiation by Negative Regulation of Transcription

Epicardin/capsulin/Pod-1, expressed in skeletal myoblasts within brachial arches and in the condensing mesenchyme, is a member of the basic helix-loop-helix (bHLH) transcription factor family that is involved in various cell differentiation processes. In this study, we examined the functional properties of epicardin/capsulin/Pod-1 in differentiation. The yeast and mammalian two-hybrid systems showed physical associations between epicardin/capsulin/Pod-1 and E2A, both of which were present in the nuclei. The bHLH domains mediated this association. Ectopic expression of epicardin/capsulin/Pod-1 inhibited E2A-dependent activation of the exogenous and endogenous expression of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) gene, and the muscle creatine kinase gene that encodes the predominant creatine kinase isoform expressed in mammalian skeletal muscle. Transfection with epicardin/capsulin/Pod-1 small interfering RNA abolished the epicardin/capsulin/Pod-1-mediated suppression of E12-dependent activation of the p21 promoter. Chromatin immunoprecipitation assay showed that epicardin/capsulin/Pod-1 was physically associated with the muscle creatine kinase promoter in vivo. Moreover, terminal differentiation of C2C12 myoblasts was inhibited by exogenous introduction of epicardin/capsulin/Pod-1. These inhibitory functions of epicardin/capsulin/Pod-1 closely resemble those of the bHLH inhibitor Twist protein. These results indicate that epicardin/capsulin/Pod-1 functions as a negative regulator of differentiation of myoblasts through transcription in at least two distinct steps, cell growth arrest and lineage-specific differentiation.