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1
Vissing, Kristian, Jesper L. Andersen, Stephen D. R. Harridge, Claudia Sandri, Andreas Hartkopp, Michael Kjaer, and Peter Schjerling. "Gene expression of myogenic factors and phenotype-specific markers in electrically stimulated muscle of paraplegics." Journal of Applied Physiology 99, no. 1 (July 2005): 164–72. http://dx.doi.org/10.1152/japplphysiol.01172.2004.
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The transcription factors myogenin and MyoD have been suggested to be involved in maintaining slow and fast muscle-fiber phenotypes, respectively, in rodents. Whether this is also the case in human muscle is unknown. To test this, 4 wk of chronic, low-frequency electrical stimulation training of the tibialis anterior muscle of paraplegic subjects were used to evoke a fast-to-slow transformation in muscle phenotype. It was hypothesized that this would result from an upregulation of myogenin and a downregulation of MyoD. The training evoked the expected mRNA increase for slow fiber-specific markers myosin heavy chain I and 3-hydroxyacyl-CoA dehydrogenase A, whereas an mRNA decrease was seen for fast fiber-specific markers myosin heavy chain IIx and glycerol phosphate dehydrogenase. Although the slow fiber-specific markers citrate synthase and muscle fatty acid binding protein did not display a significant increase in mRNA, they did tend to increase. As hypothesized, myogenin mRNA was upregulated. However, contrary to the hypothesis, MyoD mRNA also increased, although later than myogenin. The mRNA levels of the other myogenic regulatory factor family members, myogenic factor 5 and myogenic regulatory factor 4, and the myocyte enhancer factor (MEF) family members, MEF-2A and MEF-2C, did not change. The results indicate that myogenin is indeed involved in the regulation of the slow oxidative phenotype in human skeletal muscle fibers, whereas MyoD appears to have a more complex regulatory function.
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Dieli-Conwright, Christina M., Tanya M. Spektor, Judd C. Rice, Fred R. Sattler, and E. Todd Schroeder. "Influence of hormone replacement therapy on eccentric exercise induced myogenic gene expression in postmenopausal women." Journal of Applied Physiology 107, no. 5 (November 2009): 1381–88. http://dx.doi.org/10.1152/japplphysiol.00590.2009.
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Hormone replacement therapy (HRT) is used in postmenopausal women to relieve symptoms of menopause and prevent osteoporosis. We sought to evaluate changes in mRNA expression of key myogenic factors in postmenopausal women taking and not taking HRT following a high-intensity eccentric resistance exercise. Fourteen postmenopausal women were studied and included 6 control women not using HRT (59 ± 4 years, 63 ± 17 kg) and 8 women using traditional HRT (59 ± 4 yr, 89 ± 24 kg). Both groups performed 10 sets of 10 maximal eccentric repetitions of single-leg extension on a Cybex dynamometer at 60°/s. Muscle biopsies of the vastus lateralis were obtained from the exercised leg at baseline and 4 h after the exercise bout. Gene expression was determined using RT-PCR for follistatin, forkhead box 3A (FOXO3A), muscle atrophy F-box (MAFbx), muscle ring finger-1 (MuRF-1), myogenic differentiation factor (MyoD), myogenin, myostatin, myogenic factor 5 (Myf5), and muscle regulatory factor 4 (MRF4). At rest, the HRT group expressed higher levels of MyoD, myogenin, Myf5, MRF4, and follistatin ( P < 0.05). In response to eccentric exercise, follistatin, MyoD, myogenin, Myf5, and MRF4 were significantly increased ( P ≤ 0.05) and FOXO3A, MAFbx, MuRF-1, and myostatin were significantly decreased in the control and HRT groups ( P ≤ 0.05). Significantly greater changes in mRNA expression of follistatin, FOXO3A, MAFbx, MuRF-1, MyoD, myogenin, myostatin, Myf5, and MRF4 (p≤0.05) occurred in the HRT group than in the control group after exercise. These data suggest that postmenopausal women using HRT express higher myogenic regulatory factor gene expression, which may reflect an attempt to preserve muscle mass. Furthermore, postmenopausal women using HRT experienced a greater myogenic response to maximal eccentric exercise.
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Smith, T. H., N. E. Block, S. J. Rhodes, S. F. Konieczny, and J. B. Miller. "A unique pattern of expression of the four muscle regulatory factor proteins distinguishes somitic from embryonic, fetal and newborn mouse myogenic cells." Development 117, no. 3 (March 1, 1993): 1125–33. http://dx.doi.org/10.1242/dev.117.3.1125.
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A unique pattern of expression of the four muscle regulatory factor (MRF) proteins was found to distinguish early somitic from embryonic, fetal and newborn limb myogenic cells in vitro. Expression of the myosin heavy chain (MHC), MyoD, myogenin, Myf-5, and MRF4 proteins was examined by immunocytochemistry in cultures of four distinct types of mouse myogenic cells: somitic (E8.5), embryonic (E11.5), fetal (E16.5) and newborn limb. In embryonic, fetal and newborn cultures, the MRF proteins were expressed in generally similar patterns: MyoD was the first MRF expressed; MyoD and myogenin were expressed by more cells than Myf-5 or MRF4; and each of the four MRFs was found both in cells that expressed MHC and in cells that did not express MHC. In cultures of somitic cells, in contrast, Myf-5 was expressed first and by more cells than MyoD or myogenin; MRF4 was not detected; and the MRFs were never found to be coexpressed with MHC in the same cell. Thus, some somitic cells had the unexpected ability to maintain MHC expression in the absence of detectable MRF protein expression. The different myogenic programs of embryonic, fetal and newborn myogenic cells are not, therefore, a simple result of qualitatively different MRF expression patterns, whereas myogenesis by somitic cells does include a unique pattern of MRF expression.
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Block, N. E., and J. B. Miller. "Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells." Molecular and Cellular Biology 12, no. 6 (June 1992): 2484–92. http://dx.doi.org/10.1128/mcb.12.6.2484.
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Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes.
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Block, N. E., and J. B. Miller. "Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells." Molecular and Cellular Biology 12, no. 6 (June 1992): 2484–92. http://dx.doi.org/10.1128/mcb.12.6.2484-2492.1992.
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Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes.
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Marsh, Daniel R., David S. Criswell, James A. Carson, and Frank W. Booth. "Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats." Journal of Applied Physiology 83, no. 4 (October 1, 1997): 1270–75. http://dx.doi.org/10.1152/jappl.1997.83.4.1270.
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Marsh, Daniel R., David S. Criswell, James A. Carson, and Frank W. Booth. Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats. J. Appl. Physiol. 83(4): 1270–1275, 1997.—Myogenic factor mRNA expression was examined during muscle regeneration after bupivacaine injection in Fischer 344/Brown Norway F1 rats aged 3, 18, and 31 mo of age (young, adult, and old, respectively). Mass of the tibialis anterior muscle in the young rats had recovered to control values by 21 days postbupivacaine injection but in adult and old rats remained 40% less than that of contralateral controls at 21 and 28 days of recovery. During muscle regeneration, myogenin mRNA was significantly increased in muscles of young, adult, and old rats 5 days after bupivacaine injection. Subsequently, myogenin mRNA levels in young rat muscle decreased to postinjection control values by day 21 but did not return to control values in 28-day regenerating muscles of adult and old rats. The expression of MyoD mRNA was also increased in muscles at day 5 of regeneration in young, adult, and old rats, decreased to control levels by day 14 in young and adult rats, and remained elevated in the old rats for 28 days. In summary, either a diminished ability to downregulate myogenin and MyoD mRNAs in regenerating muscle occurs in old rat muscles, or the continuing myogenic effort includes elevated expression of these mRNAs.
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Di Filippo, Ester Sara, Domiziana Costamagna, Giorgia Giacomazzi, Álvaro Cortés-Calabuig, Agata Stryjewska, Danny Huylebroeck, Stefania Fulle, and Maurilio Sampaolesi. "Zeb2 Regulates Myogenic Differentiation in Pluripotent Stem Cells." International Journal of Molecular Sciences 21, no. 7 (April 5, 2020): 2525. http://dx.doi.org/10.3390/ijms21072525.
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Skeletal muscle differentiation is triggered by a unique family of myogenic basic helix-loop-helix transcription factors, including MyoD, MRF-4, Myf-5, and Myogenin. These transcription factors bind promoters and distant regulatory regions, including E-box elements, of genes whose expression is restricted to muscle cells. Other E-box binding zinc finger proteins target the same DNA response elements, however, their function in muscle development and regeneration is still unknown. Here, we show that the transcription factor zinc finger E-box-binding homeobox 2 (Zeb2, Sip-1, Zfhx1b) is present in skeletal muscle tissues. We investigate the role of Zeb2 in skeletal muscle differentiation using genetic tools and transgenic mouse embryonic stem cells, together with single-cell RNA-sequencing and in vivo muscle engraftment capability. We show that Zeb2 over-expression has a positive impact on skeletal muscle differentiation in pluripotent stem cells and adult myogenic progenitors. We therefore propose that Zeb2 is a novel myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood.
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Sengupta, Archya, Sandip Mukherjee, Shelley Bhattacharya, Samar Kumar Saha, and Ansuman Chattopadhyay. "Expression Pattern of Myogenic Regulatory Transcription Factor mRNAs in the Embryo and AdultLabeo rohita(Hamilton, 1822)." International Journal of Zoology 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/259685.
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Understanding the regulation of skeletal muscle development is important to meet the increasing demand of Indian major carpLabeo rohita. Myogenic regulatory factors (MRFs) along with myocyte specific enhancer factor 2 (MEF2) play the pivotal role in the determination and differentiation of skeletal muscle. The majority of skeletal muscle genes require both MRFs and MEF2 family members to activate their transcription. In this study, the expression pattern of MyoD, myf-5, myogenin, and MEF2A was observed from 6 h after fertilization to 12 months of age using semiquantitative RT-PCR as well as real-time PCR method. MyoD and myf-5 mRNAs were expressed at high level at the early embryonic stages. Myogenin and MEF2A were expressed after MyoD and myf-5 and remained active up to adult stage. Expression of MyoD was lower than that of Myf-5 after the 5th month. Partial sequencing of MyoD, myf-5, and MEF2A was done to draw phylogeny. In phylogenetic study,LabeoMyoD, MEF2A and myf-5 were found to be closely related to those of common carp. The present investigation suggests that the four transcription factors play pivotal role in the regulation of muscle growth ofLabeo rohitain an overlapping and interconnected way.
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Raue, Ulrika, Dustin Slivka, Bozena Jemiolo, Chris Hollon, and Scott Trappe. "Myogenic gene expression at rest and after a bout of resistance exercise in young (18–30 yr) and old (80–89 yr) women." Journal of Applied Physiology 101, no. 1 (July 2006): 53–59. http://dx.doi.org/10.1152/japplphysiol.01616.2005.
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The purpose of this study was to investigate mRNA expression of several key skeletal muscle myogenic controllers; myogenic differentiation factor (MyoD), muscle regulatory factor 4 (MRF4), myogenic factor 5 (Myf5), myogenin, myostatin, and myocyte enhancer factor 2 (MEF2) at rest and 4 h after a single bout of resistance exercise (RE) in young and old women. Eight young women (YW; 23 ± 2 yr, 67 ± 5 kg) and six old women (OW; 85 ± 1 yr, 67 ± 4 kg) performed 3 sets of 10 repetitions of bilateral knee extensions at 70% of one repetition maximum. Muscle biopsies were taken from the vastus lateralis before and 4 h after RE. Using real-time RT PCR, mRNA from the muscle samples was amplified and normalized to GAPDH. At rest, OW expressed higher ( P < 0.05) levels of MyoD, MRF4, Myf5, myogenin, and myostatin compared with YW. In response to RE, there was a main time effect ( P < 0.05) for the YW and OW combined in the upregulation of MyoD (2.0-fold) and MRF4 (1.4-fold) and in the downregulation of myostatin (2.2-fold). There was a trend ( P = 0.08) for time × age interaction in MRF4. These data show that old women express higher myogenic mRNA levels at rest. The higher resting myogenic mRNA levels in old women may reflect an attempt to preserve muscle mass and function. When challenged with RE, old women appear to respond in a similar manner as young women.
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Megeney, Lynn A., and Michael A. Rudnicki. "Determination versus differentiation and the MyoD family of transcription factors." Biochemistry and Cell Biology 73, no. 9-10 (September 1, 1995): 723–32. http://dx.doi.org/10.1139/o95-080.
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The myogenic regulatory factors (MRFs) form a family of basic helix–loop–helix transcription factors consisting of Myf-5, MyoD, myogenin, and MRF4. The MRFs play key regulatory roles in the development of skeletal muscle during embryogenesis. Sequence homology, expression patterns, and genetargeting experiments have revealed a two-tiered subclassification within the MRF family. Myf-5 and MyoD are more homologous to one another than to the others, are expressed in myoblasts before differentiation, and are required for the determination or survival of muscle progenitor cells. By contrast, myogenin and MRF4 are more homologous to one another than to the others and are expressed upon differentiation, and myogenin is required in vivo as a differentiation factor while the role of MRF4 remains unclear. On this basis, MyoD and Myf-5 are classified as primary MRFs, as they are required for the determination of myoblasts, and myogenin and MRF4 are classified as secondary MRFs, as they likely function during terminal differentiation.Key words: MyoD, Myf-5, myogenin, MRF4, skeletal muscle.
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Wyzykowski, Jeffrey C., Therry I. Winata, Natalia Mitin, Elizabeth J. Taparowsky, and Stephen F. Konieczny. "Identification of Novel MyoD Gene Targets in Proliferating Myogenic Stem Cells." Molecular and Cellular Biology 22, no. 17 (September 1, 2002): 6199–208. http://dx.doi.org/10.1128/mcb.22.17.6199-6208.2002.
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ABSTRACT A major control point for skeletal myogenesis revolves around the muscle basic helix-loop-helix gene family that includes MyoD, Myf-5, myogenin, and MRF4. Myogenin and MRF4 are thought to be essential to terminal differentiation events, whereas MyoD and Myf-5 are critical to establishing the myogenic cell lineage and producing committed, undifferentiated myogenic stem cells (myoblasts). Although mouse genetic studies have revealed the importance of MyoD and Myf-5 for myoblast development, the genetic targets of MyoD and Myf-5 activity in undifferentiated myoblasts remain unknown. In this study, we investigated the function of MyoD as a transcriptional activator in undifferentiated myoblasts. By using conditional expression of MyoD, in conjunction with suppression subtractive hybridizations, we show that the Id3 and NP1 (neuronal pentraxin 1) genes become transcriptionally active following MyoD induction in undifferentiated myoblasts. Activation of Id3 and NP1 represents a stable, heritable event that does not rely on continued MyoD activity and is not subject to negative regulation by an activated H-Ras G12V protein. These results are the first to demonstrate that MyoD functions as a transcriptional activator in myogenic stem cells and that this key myogenic regulatory factor exhibits different gene target specificities, depending upon the cellular environment.
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Braun, T., E. Bober, M. A. Rudnicki, R. Jaenisch, and H. H. Arnold. "MyoD expression marks the onset of skeletal myogenesis in Myf-5 mutant mice." Development 120, no. 11 (November 1, 1994): 3083–92. http://dx.doi.org/10.1242/dev.120.11.3083.
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The expression pattern of myogenic regulatory factors and myotome-specific contractile proteins was studied during embryonic development of Myf-5 mutant mice by in situ hybridization and immunohistochemistry. In contrast to somites in wild-type embryos, no expression of myogenin and Myf-6 (MRF4), or any other myotomal markers was detected in mutant animals at E9.0 and E10.0 indicating that Myf-5 plays a crucial role during this developmental period. Significantly, the onset of MyoD expression in rostral somites of E10.5 embryos was unaffected by the Myf-5 mutation suggesting that the activation of the MyoD gene occurs independently of Myf-5 at the correct developmental time. Immediately after the activation of MyoD myogenin transcripts and protein accumulated within the myotome. The first contractile proteins of the sarcomeric apparatus appeared slightly later. By E11.5 the expression of muscle markers were indistinguishable between wild-type and Myf-5 mutant mice. The migration of muscle precursor cells that leave the somites to form limb musculature was monitored in Myf-5-mutant mice by Pax-3 expression. Pax-3-positive cells were equally found in somites and limbs of E10.0 wild-type and mutant mice indicating that myogenic factor expression at the level of somites is not a prerequisite for determination and subsequent migration of limb precursor cells.
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Hughes, S. M., J. M. Taylor, S. J. Tapscott, C. M. Gurley, W. J. Carter, and C. A. Peterson. "Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones." Development 118, no. 4 (August 1, 1993): 1137–47. http://dx.doi.org/10.1242/dev.118.4.1137.
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Each of the myogenic helix-loop-helix transcription factors (MyoD, Myogenin, Myf-5, and MRF4) is capable of activating muscle-specific gene expression, yet distinct functions have not been ascribed to the individual proteins. We report here that MyoD and Myogenin mRNAs selectively accumulate in hindlimb muscles of the adult rat that differ in contractile properties: MyoD is prevalent in fast twitch and Myogenin in slow twitch muscles. The distribution of MyoD and Myogenin transcripts also differ within a single muscle and correlate with the proportions of fast glycolytic and slow oxidative muscle fibres, respectively. Furthermore, the expression of a transgene consisting of a muscle-specific cis-regulatory region from the myoD gene controlling lacZ was primarily associated with the fast glycolytic fibres. Alteration of the fast/slow fibre type distribution by thyroid hormone treatment or by cross-reinnervation resulted in a corresponding alteration in the MyoD/Myogenin mRNA expression pattern. These findings show that the expression of specific myogenic helix-loop-helix regulators is under the control of innervation and humoral factors and may mediate differential control of contractile protein gene expression in adult muscle.
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Jeanplong, F., JJ Bass, HK Smith, SP Kirk, R. Kambadur, M. Sharma, and JM Oldham. "Prolonged underfeeding of sheep increases myostatin and myogenic regulatory factor Myf-5 in skeletal muscle while IGF-I and myogenin are repressed." Journal of Endocrinology 176, no. 3 (March 1, 2003): 425–37. http://dx.doi.org/10.1677/joe.0.1760425.
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The IGF axis is nutritionally sensitive in vivo and IGFs stimulate myoblast proliferation and differentiation in vitro, while myostatin inhibits these processes in vitro. We hypothesised that underfeeding would reversibly inhibit the myogenic activity of satellite cells in vivo together with decreased IGF-I and increased myostatin in muscle. Satellite cell activity was measured indirectly from the expression of proliferating cell nuclear antigen (PCNA) and the myogenic regulatory factors (MRFs), MyoD, Myf-5 and myogenin. Young sheep were underfed (30% of maintenance) and some killed after 1, 4, 12, 17, 21 and 22 weeks. Remaining underfed animals were then re-fed a control ration of pellets and killed after 2 days, and 1, 6 and 30 weeks. Expression of PCNA and MRFs decreased during the first week of underfeeding. This coincided with reduced IGF-I and myostatin mRNA, and processed myostatin. Subsequently, Myf-5, MyoD, myostatin mRNA and processed myostatin increased, suggesting that satellite cells may have become progressively quiescent. Long-term underfeeding caused muscle necrosis in some animals and IGF-I and MRF expression was increased in these, indicating the activation of satellite cells for muscle repair. Re-feeding initiated rapid muscle growth and increased expression of PCNA, IGF-I and the MRFs concurrently with decreased myostatin proteins. In conclusion, these data indicate that IGF-I and myostatin may work in a coordinated manner to regulate the proliferation, differentiation and quiescence of satellite cells in vivo.
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Grabiec, K., J. Tokarska, M. Milewska, M. Błaszczyk, M. Gajewska, and K. Grzelkowska-Kowalczyk. "Interleukin-1β stimulates early myogenesis of mouse C2C12 myoblasts: the impact on myogenic regulatory factors, extracellular matrix components, IGF binding proteins and protein kinases." Polish Journal of Veterinary Sciences 16, no. 2 (June 1, 2013): 255–64. http://dx.doi.org/10.2478/pjvs-2013-0036.
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AbstractThe purpose of the study was to examine the mechanisms important for early myogenesis in mouse C2C12 myogenic cells exposed to interleukin-1β. Cyclin A and cyclin B1 were increased by interleukin-1β (1 ng/ml), but the level of cyclin D1 and total DNA content was unaffected. Fusion index and the rate of protein synthesis was increased in the presence of IL-1β, but these effects were limited to 3-day-treatment. IL-1β increased the level of MyoD, myogenin and MHC on the 3rd day of differentiation, without altering the content of the active form of myostatin, as well as it augmented the level of fibronectin, integrin β1 and full length 100 kDa form of ADAM12. IL-1β caused a decrease in IGFBP-4 and IGFBP-6 levels and a marked increase in IGFBP-5. The phosphorylation of PKB and ERK1/2 and the cellular content of p38 were elevated by IL-1β. We conclude that the myogenic effect of IL-1β was limited to the onset of myoblast fusion and was associated with: i) increase in the level of myogenic transcription factors i.e. MyoD and myogenin expression, ii) modification of extracellular matrix assembly and signaling, manifested by an increase in fibronectin, integrin-β1 and ADAM12 content, iii) drop in IGFBP-4 and IGFBP-6, and an increase in IGFBP-5, that could alter the local IGF-1 bioavailability, and iv) increase in phosphorylation of PKB and ERK1/2, and the expression of p38 kinase, leading to activation of intracellular pathways essential for myogenic differentiation.
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Husseiny, Walaa A., Abeer A. I. Hassanin, Adel A. S. El Nabtiti, Karim Khalil, and Ahmed Elaswad. "Silver Nanoparticles as Modulators of Myogenesis-Related Gene Expression in Chicken Embryos." Genes 12, no. 5 (April 22, 2021): 629. http://dx.doi.org/10.3390/genes12050629.
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The present study was conducted to investigate the effects of colloidal nanoparticles of silver (Nano-Ag) on the expression of myogenesis-related genes in chicken embryos. The investigated genes included the members of the myogenic regulatory factors family (MRFs) and myocyte enhancer factor 2A (MEF2A) genes. A total of 200 fertilized broiler eggs (Indian River) were randomly distributed into four groups; non-injected control, injected control with placebo, treatment I in ovo injected with 20 ppm Nano-Ag, and treatment II in ovo injected with 40 ppm Nano-Ag. The eggs were then incubated for 21 days at the optimum temperature and humidity conditions. Breast muscle tissues were collected at the 5th, 8th, and 18th days of the incubation period. The mRNA expression of myogenic determination factor 1 (MYOD1), myogenic factor 5 (MYF5), myogenic factor 6 (MYF6), myogenin (MYOG), and MEF2A was measured at the three sampling points using real-time quantitative PCR, while MYOD1 protein expression was evaluated on day 18 using western blot. Breast muscle tissues were histologically examined on day 18 to detect the changes at the cellular level. Our results indicate that myogenesis was enhanced with the low concentration (20 ppm) of Nano-Ag due to the higher expression of MYOD1, MYF5, and MYF6 at the transcriptional level and MYOD1 at the translational level. Moreover, histological analysis revealed the presence of hyperplasia (31.4% more muscle fibers) in treatment I (injected with 20 ppm). Our findings indicate that in ovo injection of 20 ppm Nano-Ag enhances the development of muscles in chicken embryos compared with the 40-ppm dosage and provide crucial information for the use of silver nanoparticles in poultry production.
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Kanzleiter, Timo, Markus Jähnert, Gunnar Schulze, Joachim Selbig, Nicole Hallahan, Robert Wolfgang Schwenk, and Annette Schürmann. "Exercise training alters DNA methylation patterns in genes related to muscle growth and differentiation in mice." American Journal of Physiology-Endocrinology and Metabolism 308, no. 10 (May 15, 2015): E912—E920. http://dx.doi.org/10.1152/ajpendo.00289.2014.
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The adaptive response of skeletal muscle to exercise training is tightly controlled and therefore requires transcriptional regulation. DNA methylation is an epigenetic mechanism known to modulate gene expression, but its contribution to exercise-induced adaptations in skeletal muscle is not well studied. Here, we describe a genome-wide analysis of DNA methylation in muscle of trained mice ( n = 3). Compared with sedentary controls, 2,762 genes exhibited differentially methylated CpGs ( P < 0.05, meth diff >5%, coverage >10) in their putative promoter regions. Alignment with gene expression data ( n = 6) revealed 200 genes with a negative correlation between methylation and expression changes in response to exercise training. The majority of these genes were related to muscle growth and differentiation, and a minor fraction involved in metabolic regulation. Among the candidates were genes that regulate the expression of myogenic regulatory factors ( Plexin A2) as well as genes that participate in muscle hypertrophy ( Igfbp4) and motor neuron innervation ( Dok7). Interestingly, a transcription factor binding site enrichment study discovered significantly enriched occurrence of CpG methylation in the binding sites of the myogenic regulatory factors MyoD and myogenin. These findings suggest that DNA methylation is involved in the regulation of muscle adaptation to regular exercise training.
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Tan, Xungang, Yuqing Zhang, Pei-Jun Zhang, Peng Xu, and Yongli Xu. "Molecular structure and expression patterns of flounder (Paralichthys olivaceus) Myf-5, a myogenic regulatory factor." Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 145, no. 2 (October 2006): 204–13. http://dx.doi.org/10.1016/j.cbpb.2006.07.008.
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Johnston, I. A., H. A. McLay, M. Abercromby, and D. Robins. "Phenotypic plasticity of early myogenesis and satellite cell numbers in atlantic salmon spawning in upland and lowland tributaries of a river system." Journal of Experimental Biology 203, no. 17 (September 1, 2000): 2539–52. http://dx.doi.org/10.1242/jeb.203.17.2539.
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Early myogenesis was studied in the offspring of Atlantic salmon (Salmo salar L.) spawning in a lowland (Sheeoch) and an upland (Baddoch) tributary of the River Dee System, Aberdeenshire, Scotland. Eggs from each population were incubated at the simulated natural thermal regimes of each stream, which was on average 2.8 degrees C cooler for the Baddoch than for the Sheeoch. Relationships between muscle cellularity variables, the density of myonuclei and responses to temperature were investigated using multivariate statistical techniques. These revealed highly significant temperature effects (P<0.001) at hatch (H) and first feeding (FF) and significant interactions between population and temperature (P<0.001), indicating that Baddoch and Sheeoch salmon responded differently to the two temperature regimes. The total cross-sectional area of white muscle (WF.ta) at the adipose fin was relatively independent of temperature at hatch and first feeding in the Sheeoch population. In contrast, for alevins of Baddoch origin, WF.ta was 18.9% (H) and 30.5% (FF) higher in fish incubated at Baddoch than at Sheeoch temperatures. At hatch, there were 15.6% more white muscle fibres (WF.no) at the cooler incubation temperature in fish of Sheeoch origin and 6.0% more in fish of Baddoch origin. However, by first feeding, the difference in WF.no between temperatures had narrowed to 7.2% in the Sheeoch fish and increased to 17.4% in the Baddoch population. In contrast, at hatch, the density of myonuclei was 59.8% higher at the warmer incubation temperature in the Sheeoch population and 23.5% higher in the Baddoch population, but differences were less evident at first feeding. In Baddoch fish, 22.5% of the total muscle nuclei were actively dividing at first feeding, as assessed by staining for proliferating cell nuclear antigen (PCNA). Of the PCNA-positive nuclei, 78% were present in cells that stained for the c-met tyrosine kinase receptor, a marker of satellite cells and their division products. The proportion of c-met-positive cells staining for individual myogenic regulatory factors was 72.4% for the myogenic transcription factor MyoD, 76.3% for the myogenic transcription factor Myf-5, 62.1% for myogenin and 48.7% for the myogenic transcription factor Myf-6. For the Sheeoch population, there were 26.5% more c-met-expressing (P<0.01) and 23.2% more myogenic-regulatory-factor-expressing (P<0.05) cells at Sheeoch than at Baddoch temperatures. In contrast, incubation temperature had no significant effects on satellite cell density in the Baddoch population.
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Zhou, Hongyi, Huabo Su, and Weiqin Chen. "Neddylation Regulates Class IIa and III Histone Deacetylases to Mediate Myoblast Differentiation." International Journal of Molecular Sciences 22, no. 17 (September 1, 2021): 9509. http://dx.doi.org/10.3390/ijms22179509.
Full textAbstract:
As the largest tissue in the body, skeletal muscle has multiple functions in movement and energy metabolism. Skeletal myogenesis is controlled by a transcriptional cascade including a set of muscle regulatory factors (MRFs) that includes Myogenic Differentiation 1 (MYOD1), Myocyte Enhancer Factor 2 (MEF2), and Myogenin (MYOG), which direct the fusion of myogenic myoblasts into multinucleated myotubes. Neddylation is a posttranslational modification that covalently conjugates ubiquitin-like NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to protein targets. Inhibition of neddylation impairs muscle differentiation; however, the underlying molecular mechanisms remain less explored. Here, we report that neddylation is temporally regulated during myoblast differentiation. Inhibition of neddylation through pharmacological blockade using MLN4924 (Pevonedistat) or genetic deletion of NEDD8 Activating Enzyme E1 Subunit 1 (NAE1), a subunit of the E1 neddylation-activating enzyme, blocks terminal myoblast differentiation partially through repressing MYOG expression. Mechanistically, we found that neddylation deficiency enhances the mRNA and protein expressions of class IIa histone deacetylases 4 and 5 (HDAC4 and 5) and prevents the downregulation and nuclear export of class III HDAC (NAD-Dependent Protein Deacetylase Sirtuin-1, SIRT1), all of which have been shown to repress MYOD1-mediated MYOG transcriptional activation. Together, our findings for the first time identify the crucial role of neddylation in mediating class IIa and III HDAC co-repressors to control myogenic program and provide new insights into the mechanisms of muscle disease and regeneration.
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Kong, Y., S. E. Johnson, E. J. Taparowsky, and S. F. Konieczny. "Ras p21Val inhibits myogenesis without altering the DNA binding or transcriptional activities of the myogenic basic helix-loop-helix factors." Molecular and Cellular Biology 15, no. 10 (October 1995): 5205–13. http://dx.doi.org/10.1128/mcb.15.10.5205.
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MRF4, MyoD, myogenin, and Myf-5 are muscle-specific basic helix-loop-helix transcription factors that share the ability to activate the expression of skeletal muscle genes such as those encoding alpha-actin, myosin heavy chain, and the acetylcholine receptor subunits. The muscle regulatory factors (MRFs) also exhibit the unique capacity to initiate the myogenic program when ectopically expressed in a variety of nonmuscle cell types, most notably C3H10T1/2 fibroblasts (10T1/2 cells). The commitment of myoblasts to terminal differentiation, although positively regulated by the MRFs, also is controlled negatively by a variety of agents, including several growth factors and oncoproteins such as fibroblast growth factor (FGF-2), transforming growth factor beta 1 (TGF-beta 1), and Ras p21Val. The molecular mechanisms by which these varied agents alter myogenic terminal differentiation events remain unclear. In an effort to establish whether Ras p21Val represses MRF activity by directly targeting the MRF proteins, we examined the DNA binding and transcription activation potentials of MRF4 and MyoD when expressed in 10T1/2 cells or in 10T1/2 cells expressing Ras p21Val. Our results demonstrate that Ras p21Val inhibits terminal differentiation events by targeting the basic domain of the MRFs, and yet the mechanism underlying this inhibition does not involve altering the DNA binding or the inherent transcriptional activity of these regulatory factors. In contrast, FGF-2 and TGF-beta 1 block terminal differentiation by repressing the transcriptional activity of the MRFs. We conclude that the Ras p21Val block in differentiation operates via an intracellular signaling pathway that is distinct from the FGF-2 and TGF-beta 1 pathways.
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Mueller, Adam C., Magdalena A. Cichewicz, Bijan K. Dey, Ryan Layer, Brian J. Reon, Jeffrey R. Gagan, and Anindya Dutta. "MUNC, a Long Noncoding RNA That Facilitates the Function of MyoD in Skeletal Myogenesis." Molecular and Cellular Biology 35, no. 3 (November 17, 2014): 498–513. http://dx.doi.org/10.1128/mcb.01079-14.
Full textAbstract:
Anin silicoscreen for myogenic long noncoding RNAs (lncRNAs) revealed nine lncRNAs that are upregulated more than 10-fold in myotubes versus levels in myoblasts. One of these lncRNAs, MyoD upstream noncoding (MUNC, also known as DRReRNA), is encoded 5 kb upstream of the transcription start site ofMyoD, a myogenic transcription factor gene. MUNC is specifically expressed in skeletal muscle and exists as in unspliced and spliced isoforms, and its 5′ end overlaps with thecis-acting distal regulatory region (DRR) ofMyoD. Small interfering RNA (siRNA) of MUNC reduced myoblast differentiation and specifically reduced the association of MyoD to the DRR enhancer and myogenin promoter but not to another MyoD-dependent enhancer. Stable overexpression of MUNC from a heterologous promoter increased endogenousMyoD,Myogenin, andMyh3(myosin heavy chain, [MHC] gene) mRNAs but not the cognate proteins, suggesting that MUNC can act intransto promote gene expression but that this activity does not require an induction of MyoD protein. MUNC also stimulates the transcription of other genes that are not recognized as MyoD-inducible genes. Knockdown of MUNCin vivoimpaired murine muscle regeneration, implicating MUNC in primary satellite cell differentiation in the animal. We also discovered a human MUNC that is induced during differentiation of myoblasts and whose knockdown decreases differentiation, suggesting an evolutionarily conserved role of MUNC lncRNA in myogenesis. Although MUNC overlaps with the DRR enhancer, our results suggest that MUNC is not a classiccis-acting enhancer RNA (e-RNA) acting exclusively by stimulating the neighboringMyoDgene but more like a promyogenic lncRNA that acts directly or indirectly on multiple promoters to increase myogenic gene expression.
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Belaguli, Narasimhaswamy S., Wei Zhou, Thuy-Hanh T. Trinh, Mark W. Majesky, and Robert J. Schwartz. "Dominant Negative Murine Serum Response Factor: Alternative Splicing within the Activation Domain Inhibits Transactivation of Serum Response Factor Binding Targets." Molecular and Cellular Biology 19, no. 7 (July 1, 1999): 4582–91. http://dx.doi.org/10.1128/mcb.19.7.4582.
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ABSTRACT Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRFΔ5 to SRF. Increased levels of SRFΔ5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRFΔ5 variant relative to wild-type SRF. SRFΔ5 forms DNA binding-competent homodimers and heterodimers. SRFΔ5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal α-actin, cardiac α-actin, smooth α-actin, SM22α, and SRF promoter-luciferase reporter activities. Expression of SRFΔ5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal α-actin and myogenin mRNAs. SRFΔ5 repressed the serum-induced activity of the c-fos serum response element. SRFΔ5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRFΔ5, may play an important regulatory role in modulating SRF-dependent gene expression.
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Liao, Shengfa F., Zhongyue Yang, M. Shamimul Hasan, Rebecca Humphrey, Jean Feugang, Derris Burnett, and John K. Htoo. "200 Reduced growth performance of pigs fed methionine deficient diet may be associated with their reduced muscle cell differentiation." Journal of Animal Science 98, Supplement_3 (November 2, 2020): 70. http://dx.doi.org/10.1093/jas/skaa054.125.
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Abstract Methionine (Met) is the second or third limiting AA in typical swine diets and plays important roles in promoting the growth, especially, the muscle growth of pigs. This research studied the effects of dietary Met restriction on growth performance and myogenic gene expression in growing pigs. Eight genes in two families, including myogenic regulatory factor family comprising myogenic differentiation 1 (MyoD1), myogenic factor 5 (Myf5), myogenin (MyoG) and myogenic factor 6 (Myf6), and myocyte enhancer factor 2 (Mef2) family comprising Mef2A, Mef2B, Mef2C and Mef2D, were selected for analysis. Individually penned barrows (crossbred, 23.6±2.4 kg) were randomly assigned to two dietary treatments. A basal diet (Diet 1; Met-deficient) was formulated to contain 0.22% standardized ileal digestible (SID) Met and 0.52% SID Met+Cys but to meet the NRC (2012) recommendations for other nutrients. Crystalline DL-Met was added to the basal diet to generate Diet 2 adequate in Met (0.37% SID Met, 0.67% SID Met+Cys). During the four-week ad libitum feeding trial, ADG, ADFI and G:F were measured. Immediately before and after the trial, muscle samples were collected from the longissimus dorsi using a standard biopsy protocol. Total RNA was extracted from the muscle samples (TRIzol Reagent; Invitrogen) and subjected to cDNA reverse-transcription (QuantiTect Reverse Transcription Kit; Qiagen). Transcribed cDNA were used for qPCR analysis (Rotor-Gene Q System; Qiagen). Comparative ΔΔCT method was used for quantitation. Data were analyzed with Student’s T-test. Pigs fed Diet 1 (vs. Diet 2) had a lower ADG and G:F (P< 0.01). Before the feeding trial, all the tested genes had comparable mRNA levels between the two treatments (P >0.17). After the trial, Diet 1 pigs showed tendency for lower levels of Myf6 and Mef2D mRNA (P< 0.09). These results suggest that the reduced ADG and G:F is associated with the possibly-reduced muscle cell differentiation in pigs fed Met-deficient diet.
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Kobiyama, A., Y. Nihei, Y. Hirayama, K. Kikuchi, H. Suetake, IA Johnston, and S. Watabe. "Molecular cloning and developmental expression patterns of the MyoD and MEF2 families of muscle transcription factors in the carp." Journal of Experimental Biology 201, no. 20 (October 15, 1998): 2801–13. http://dx.doi.org/10.1242/jeb.201.20.2801.
Full textAbstract:
cDNA clones encoding the myogenic regulatory factors (MRFs) myogenin, MyoD and myf-5 were isolated by reverse-transcription polymerase chain reaction from larvae and embryos of the common carp (Cyprinus carpio L.). Myocyte-specific enhancer factor 2 (MEF2) cDNAs were identified from a cDNA library from adult carp. Northern blot analysis showed that MyoD, myf-5 and MEF2C transcripts were present in three-somite embryos, whereas myogenin and MEF2A transcripts were not detected until the 15-somite stage. Intense signals of myogenin and MyoD transcripts were observed even in 1-month-old juveniles. Levels of MyoD, myogenin and MEF2A transcripts declined between 1 and 7 months after hatching, and myf-5 gave only a weak signal in the oldest fish. In contrast, levels of MEF2C transcripts were considerably higher in 7-month-old juveniles than in 1-month-old larvae. mRNAs encoding carp myosin heavy chain and -actin were first detected at approximately the time of the first heartbeat, and levels were maximal in juveniles 1 month post-hatching. The relatively high levels of MRF mRNA in juvenile fish probably reflect the recruitment of new muscle fibres from the satellite cell population. It was concluded that the relative importance of the different members of the MyoD and MEF2 families of transcription factors for muscle differentiation changes during ontogeny in the carp.
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McLellan, A. S., T. Kealey, and K. Langlands. "An E box in the exon 1 promoter regulates insulin-like growth factor-I expression in differentiating muscle cells." American Journal of Physiology-Cell Physiology 291, no. 2 (August 2006): C300—C307. http://dx.doi.org/10.1152/ajpcell.00345.2005.
Full textAbstract:
Insulin-like growth factor (IGF)-I expression is subject to complex temporal and spatial regulation. Endocrine synthesis occurs in the liver, where transcription is initiated from promoters located in either exon 1 (P1) or in exon 2 (P2), whereas local transcription is mainly initiated from P1. IGF-I is expressed in a range of tissues and, in particular, is an important regulator of skeletal muscle mass, although the mechanisms of tissue-specific regulation remain to be fully characterized. Gene regulation in skeletal muscle is associated with the E box DNA element (5′-CANNTG-3′) recognized by myogenic regulatory factors (MRFs), such as MyoD1. Transcription element profiling identified a hypothetical myogenic E box (sequence 5′-CAGCTG-3′) within P1, immediately upstream of the major muscle transcriptional start site, and we sought to test its activity in differentiating C2C12 myoblasts. We found P1-driven IGF-I mRNA expression to be associated with myogenic differentiation and, moreover, that a single base-pair mutation in the E box specifically reduced expression in myofibers. A synthetic enhancer construct containing a triplet repeat of the E box was active in muscle cells and strongly induced in myofibers. The capacity of a double-stranded IGF-I E box probe (but not one bearing a single-base pair alteration) to bind C2C12 nuclear lysates increased with myogenesis, and a transactivation assay demonstrated that the E box was recognized by E protein-MRF heterodimers. Mechanisms of tissue-specific gene activation are of increasing biological interest, and we have identified a cis-element able to direct muscle-specific IGF-I gene expression.
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Kosek, David J., Jeong-su Kim, John K. Petrella, James M. Cross, and Marcas M. Bamman. "Efficacy of 3 days/wk resistance training on myofiber hypertrophy and myogenic mechanisms in young vs. older adults." Journal of Applied Physiology 101, no. 2 (August 2006): 531–44. http://dx.doi.org/10.1152/japplphysiol.01474.2005.
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Resistance training (RT) has shown the most promise in reducing/reversing effects of sarcopenia, although the optimum regime specific for older adults remains unclear. We hypothesized myofiber hypertrophy resulting from frequent (3 days/wk, 16 wk) RT would be impaired in older (O; 60–75 yr; 12 women, 13 men), sarcopenic adults compared with young (Y; 20–35 yr; 11 women, 13 men) due to slowed repair/regeneration processes. Myofiber-type distribution and cross-sectional area (CSA) were determined at 0 and 16 wk. Transcript and protein levels of myogenic regulatory factors (MRFs) were assessed as markers of regeneration at 0 and 24 h postexercise, and after 16 wk. Only Y increased type I CSA 18% ( P < 0.001). O showed smaller type IIa (−16%) and type IIx (−24%) myofibers before training ( P < 0.05), with differences most notable in women. Both age groups increased type IIa (O, 16%; Y, 25%) and mean type II (O, 23%; Y, 32%) size ( P < 0.05). Growth was generally most favorable in young men. Percent change scores on fiber size revealed an age × gender interaction for type I fibers ( P < 0.05) as growth among Y (25%) exceeded that of O (4%) men. Myogenin and myogenic differentiation factor D (MyoD) mRNAs increased ( P < 0.05) in Y and O, whereas myogenic factor (myf)-5 mRNA increased in Y only ( P < 0.05). Myf-6 protein increased ( P < 0.05) in both Y and O. The results generally support our hypothesis as 3 days/wk training led to more robust hypertrophy in Y vs. O, particularly among men. However, this differential hypertrophy adaptation was not explained by age variation in MRF expression.
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Ma, Kun, Con Mallidis, Jorge Artaza, Wayne Taylor, Nestor Gonzalez-Cadavid, and Shalender Bhasin. "Characterization of 5′-regulatory region of human myostatin gene: regulation by dexamethasone in vitro." American Journal of Physiology-Endocrinology and Metabolism 281, no. 6 (December 1, 2001): E1128—E1136. http://dx.doi.org/10.1152/ajpendo.2001.281.6.e1128.
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We cloned and characterized a 3.3-kb fragment containing the 5′-regulatory region of the human myostatin gene. The promoter sequence contains putative muscle growth response elements for glucocorticoid, androgen, thyroid hormone, myogenic differentiation factor 1, myocyte enhancer factor 2, peroxisome proliferator-activated receptor, and nuclear factor-κB. To identify sites important for myostatin's gene transcription and regulation, eight deletion constructs were placed in C2C12 and L6 skeletal muscle cells. Transcriptional activity of the constructs was found to be significantly higher in myotubes compared with that of myoblasts. To investigate whether glucocorticoids regulate myostatin gene expression, we incubated both cell lines with dexamethasone. On both occasions, dexamethasone dose dependently increased both the promoter's transcriptional activity and the endogenous myostatin expression. The effects of dexamethasone were blocked when the cells were coincubated with the glucocorticoid receptor antagonist RU-486. These findings suggest that glucocorticoids upregulate myostatin expression by inducing gene transcription, possibly through a glucocorticoid receptor-mediated pathway. We speculate that glucocorticoid-associated muscle atrophy might be due in part to the upregulation of myostatin expression.
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Vissing, Kristian, Sean L. McGee, Carsten Roepstorff, Peter Schjerling, Mark Hargreaves, and Bente Kiens. "Effect of sex differences on human MEF2 regulation during endurance exercise." American Journal of Physiology-Endocrinology and Metabolism 294, no. 2 (February 2008): E408—E415. http://dx.doi.org/10.1152/ajpendo.00403.2007.
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Women exhibit an enhanced capability for lipid metabolism during endurance exercise compared with men. The underlying regulatory mechanisms behind this sex-related difference are not well understood but may comprise signaling through a myocyte enhancer factor 2 (MEF2) regulatory pathway. The primary purpose of this study, therefore, was to investigate the protein signaling of MEF2 regulatory pathway components at rest and during 90 min of bicycling exercise at 60% V̇o2peak in healthy, moderately trained men ( n = 8) and women ( n = 9) to elucidate the potential role of these proteins in substrate utilization during exercise. A secondary purpose was to screen for mRNA expression of MEF2 isoforms and myogenic regulatory factor (MRF) family members of transcription factors at rest and during exercise. Muscle biopsies were obtained before and immediately after exercise. Nuclear AMP-activated protein kinase-α (αAMPK) Thr172 ( P < 0.001), histone deacetylase 5 (HDAC5) Ser498 ( P < 0.001), and MEF2 Thr ( P < 0.01) phosphorylation increased with exercise. No significant sex differences were observed at rest or during exercise. At rest, no significant sex differences were observed in mRNA expression of the measured transcription factors. mRNA for transcription factors MyoD, myogenin, MRF4, MEF2A, MEF2C, MEF2D, and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) were significantly upregulated by exercise. Of these, MEF2A mRNA increased 25% specifically in women ( P < 0.05), whereas MEF2D mRNA tended to increase in men ( P = 0.11). Although minor sex differences in mRNA expression were observed, the main finding of the present study was the implication of a joint signaling action of AMPK, HDAC5, and PGC1α on MEF2 in the immediate regulatory response to endurance exercise. This signaling response was independent of sex.
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Dominov, Janice A., Jonathan J. Dunn, and Jeffrey Boone Miller. "Bcl-2 Expression Identifies an Early Stage of Myogenesis and Promotes Clonal Expansion of Muscle Cells." Journal of Cell Biology 142, no. 2 (July 27, 1998): 537–44. http://dx.doi.org/10.1083/jcb.142.2.537.
Full textAbstract:
We show that Bcl-2 expression in skeletal muscle cells identifies an early stage of the myogenic pathway, inhibits apoptosis, and promotes clonal expansion. Bcl-2 expression was limited to a small proportion of the mononucleate cells in muscle cell cultures, ranging from ∼1–4% of neonatal and adult mouse muscle cells to ∼5–15% of the cells from the C2C12 muscle cell line. In rapidly growing cultures, some of the Bcl-2–positive cells coexpressed markers of early stages of myogenesis, including desmin, MyoD, and Myf-5. In contrast, Bcl-2 was not expressed in multinucleate myotubes or in those mononucleate myoblasts that expressed markers of middle or late stages of myogenesis, such as myogenin, muscle regulatory factor 4 (MRF4), and myosin. The small subset of Bcl-2–positive C2C12 cells appeared to resist staurosporine-induced apoptosis. Furthermore, though myogenic cells from genetically Bcl-2–null mice formed myotubes normally, the muscle colonies produced by cloned Bcl-2–null cells contained only about half as many cells as the colonies produced by cells from wild-type mice. This result suggests that, during clonal expansion from a muscle progenitor cell, the number of progeny obtained is greater when Bcl-2 is expressed.
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Mak, K. L., R. Q. To, Y. Kong, and S. F. Konieczny. "The MRF4 activation domain is required to induce muscle-specific gene expression." Molecular and Cellular Biology 12, no. 10 (October 1992): 4334–46. http://dx.doi.org/10.1128/mcb.12.10.4334.
Full textAbstract:
MRF4 is a member of the basic helix-loop-helix muscle regulatory factor family that also includes MyoD, myogenin, and Myf-5. Overexpression of MRF4 or the other muscle regulatory factors in fibroblasts converts the cells to differentiated muscle fibers and transcriptionally activates expression of endogenous and cotransfected muscle genes. Although these factors induce a similar phenotype, they also exhibit some distinct biological activities. For example, MyoD trans activates alpha-actin and troponin I reporter genes to very high levels, whereas MRF4 efficiently activates only alpha-actin expression. Since these proteins have a common basic helix-loop-helix domain, it is likely that portions of the proteins outside of this region impart some specificity to the activity of each muscle regulatory factor. As an initial step in determining the mechanism by which MRF4 and MyoD activate gene transcription, the transcriptional activation domain of MRF4 has been characterized. Experiments utilizing chimeric proteins containing the yeast GAL4 DNA-binding domain and portions of the MRF4 protein indicate that the MRF4 activation domain is located within amino acids 10 to 30. This amino terminus is both necessary and sufficient to elicit a transcriptional response in transfected cells. The MRF4 activation domain and the related amino-terminal MyoD activation domain are capable of substituting for one another in converting fibroblasts to a myogenic phenotype and in activating expression of an alpha-actin reporter gene, although the MRF4 and MyoD activation domains on these chimeric proteins also dictate the specificity of transcriptional activation. The different primary amino acid sequences of these regions leave open the possibility that different coregulator proteins interact with the muscle regulatory factors to elicit their correct transcriptional activity during skeletal muscle development.
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Mak, K. L., R. Q. To, Y. Kong, and S. F. Konieczny. "The MRF4 activation domain is required to induce muscle-specific gene expression." Molecular and Cellular Biology 12, no. 10 (October 1992): 4334–46. http://dx.doi.org/10.1128/mcb.12.10.4334-4346.1992.
Full textAbstract:
MRF4 is a member of the basic helix-loop-helix muscle regulatory factor family that also includes MyoD, myogenin, and Myf-5. Overexpression of MRF4 or the other muscle regulatory factors in fibroblasts converts the cells to differentiated muscle fibers and transcriptionally activates expression of endogenous and cotransfected muscle genes. Although these factors induce a similar phenotype, they also exhibit some distinct biological activities. For example, MyoD trans activates alpha-actin and troponin I reporter genes to very high levels, whereas MRF4 efficiently activates only alpha-actin expression. Since these proteins have a common basic helix-loop-helix domain, it is likely that portions of the proteins outside of this region impart some specificity to the activity of each muscle regulatory factor. As an initial step in determining the mechanism by which MRF4 and MyoD activate gene transcription, the transcriptional activation domain of MRF4 has been characterized. Experiments utilizing chimeric proteins containing the yeast GAL4 DNA-binding domain and portions of the MRF4 protein indicate that the MRF4 activation domain is located within amino acids 10 to 30. This amino terminus is both necessary and sufficient to elicit a transcriptional response in transfected cells. The MRF4 activation domain and the related amino-terminal MyoD activation domain are capable of substituting for one another in converting fibroblasts to a myogenic phenotype and in activating expression of an alpha-actin reporter gene, although the MRF4 and MyoD activation domains on these chimeric proteins also dictate the specificity of transcriptional activation. The different primary amino acid sequences of these regions leave open the possibility that different coregulator proteins interact with the muscle regulatory factors to elicit their correct transcriptional activity during skeletal muscle development.
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Vélez, Emilio J., Sheida Azizi, Esmail Lutfi, Encarnación Capilla, Alberto Moya, Isabel Navarro, Jaume Fernández-Borràs, Josefina Blasco, and Joaquim Gutiérrez. "Moderate and sustained exercise modulates muscle proteolytic and myogenic markers in gilthead sea bream (Sparus aurata)." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 312, no. 5 (May 1, 2017): R643—R653. http://dx.doi.org/10.1152/ajpregu.00308.2016.
Full textAbstract:
Swimming activity primarily accelerates growth in fish by increasing protein synthesis and energy efficiency. The role of muscle in this process is remarkable and especially important in teleosts, where muscle represents a high percentage of body weight and because many fish species present continuous growth. The aim of this work was to characterize the effects of 5 wk of moderate and sustained swimming in gene and protein expression of myogenic regulatory factors, proliferation markers, and proteolytic molecules in two muscle regions (anterior and caudal) of gilthead sea bream fingerlings. Western blot results showed an increase in the proliferation marker proliferating cell nuclear antigen (PCNA), proteolytic system members calpain 1 and cathepsin D, as well as vascular endothelial growth factor protein expression. Moreover, quantitative real-time PCR data showed that exercise increased the gene expression of proteases (calpains, cathepsins, and members of the ubiquitin-proteasome system in the anterior muscle region) and the gene expression of the proliferation marker PCNA and the myogenic factor MyoD in the caudal area compared with control fish. Overall, these data suggest a differential response of the two muscle regions during swimming adaptation, with tissue remodeling and new vessel formation occurring in the anterior muscle and enhanced cell proliferation and differentiation occurring in the caudal area. In summary, the present study contributes to improving the knowledge of the role of proteolytic molecules and other myogenic factors in the adaptation of muscle to moderate sustained swimming in gilthead sea bream.
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Chen, Shuen-Ei, Eric Gerken, Yingmin Zhang, Mei Zhan, Raja K. Mohan, Andrew S. Li, Michael B. Reid, and Yi-Ping Li. "Role of TNF-α signaling in regeneration of cardiotoxin-injured muscle." American Journal of Physiology-Cell Physiology 289, no. 5 (November 2005): C1179—C1187. http://dx.doi.org/10.1152/ajpcell.00062.2005.
Full textAbstract:
Recent data suggest a physiological role for the proinflammatory cytokine TNF-α in skeletal muscle regeneration. However, the underlying mechanism is not understood. In the present study, we analyzed TNF-α-activated signaling pathways involved in myogenesis in soleus muscle injured by cardiotoxin (CTX) in TNF-α receptor double-knockout mice (p55−/−p75−/−). We found that activation of p38MAPK, which is critical for myogenesis, was blocked in CTX-injured p55−/−p75−/−soleus on day 3 postinjury when myogenic differentiation was being initiated, while activation of ERK1/2 and JNK MAPK, as well as transcription factor NF-κB, was not reduced. Consequently, the phosphorylation of transcription factor myocyte enhancer factor-2C, which is catalyzed by p38 and crucial for the expression of muscle-specific genes, was blunted. Meanwhile, expression of p38-dependent differentiation marker myogenin and p21 were suppressed. In addition, expression of cyclin D1 was fivefold that in wild-type (WT) soleus. These results suggest that myogenic differentiation is blocked or delayed in the absence of TNF-α signaling. Histological studies revealed abnormalities in regenerating p55−/−p75−/−soleus. On day 5 postinjury, new myofiber formation was clearly observed in WT soleus but not in p55−/−p75−/−soleus. To the contrary, p55−/−p75−/−soleus displayed renewed inflammation and dystrophic calcification. On day 12 postinjury, the muscle architecture of WT soleus was largely restored. Yet, in p55−/−p75−/−soleus, multifocal areas of inflammation, myofiber death, and myofibers with smaller cross-sectional area were observed. Functional studies demonstrated an attenuated recovery of contractile force in injured p55−/−p75−/−soleus. These data suggest that TNF-α signaling plays a critical regulatory role in muscle regeneration.
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Belaguli, Narasimhaswamy S., Jorge L. Sepulveda, Vishal Nigam, Frédéric Charron, Mona Nemer, and Robert J. Schwartz. "Cardiac Tissue Enriched Factors Serum Response Factor and GATA-4 Are Mutual Coregulators." Molecular and Cellular Biology 20, no. 20 (October 15, 2000): 7550–58. http://dx.doi.org/10.1128/mcb.20.20.7550-7558.2000.
Full textAbstract:
ABSTRACT Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal α-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.
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Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. "Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’." Journal of Cell Science 111, no. 6 (March 15, 1998): 769–79. http://dx.doi.org/10.1242/jcs.111.6.769.
Full textAbstract:
When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors. The differentiating part of the cell population followed a series of well-described events, including expression of myogenin, p21(WAF1), and contractile proteins, permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells did not initiate any of these events. Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression, which had been originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells were isolated and returned to the growth conditions, they progressed through the cell cycle and regained MyoD expression. These cells demonstrated identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture were designated ‘reserve cells’. Upon serum deprivation, MyoD expression rapidly decreased as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells expressed myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells did not differentiate and became the reserve cells. We also demonstrated that ectopic expression of MyoD converted the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells.
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Smith, T. H., A. M. Kachinsky, and J. B. Miller. "Somite subdomains, muscle cell origins, and the four muscle regulatory factor proteins." Journal of Cell Biology 127, no. 1 (October 1, 1994): 95–105. http://dx.doi.org/10.1083/jcb.127.1.95.
Full textAbstract:
We show by immunohistology that distinct expression patterns of the four muscle regulatory factor (MRF) proteins identify subdomains of mouse somites. Myf-5 and MyoD are, at specific stages, each expressed in both myotome and dermatome cells. Myf-5 expression is initially restricted to dorsal cells in all somites, as is MyoD expression in neck somites. In trunk somites, however, MyoD is initially expressed in ventral cells. Myogenin and MRF4 are restricted to myotome cells, though the MRF4-expressing cells are initially less widely distributed than the myogenin-expressing cells, which are at all stages found throughout the myotome. All somitic myocytes express one or more MRFs. The transiently distinct expression patterns of the four MRF proteins identify dorsal and ventral subdomains of somites, and suggest that skeletal muscle cells in somites originate at multiple sites and via multiple molecular pathways.
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Eppley, Z. A., J. Kim, and B. Russell. "A myogenic regulatory gene, qmf1, is expressed by adult myonuclei after injury." American Journal of Physiology-Cell Physiology 265, no. 2 (August 1, 1993): C397—C405. http://dx.doi.org/10.1152/ajpcell.1993.265.2.c397.
Full textAbstract:
Myogenic regulatory factors (MRFs) induce differentiation in developing muscle. We examined the role of MRFs in the repair of adult muscle using a model of stretch-induced injury in 5-wk-old chickens. The anterior latissimus dorsi muscle was stretched by loading the wing with 10% of body weight, while the contralateral muscle served as a control. At various intervals (0.5-72 h), chickens were killed by CO2 asphyxiation and the muscles were frozen. Slot hybridizations showed that the onset of high qmf1 expression occurred as early as 0.5 h, which was before regenerative processes involving satellite cell proliferation were observed. Maximal qmf1 expression varied among animals from 3 to 16 h and returned to control levels by 72 h. Within a muscle, in situ hybridization showed that maximal qmf1 expression varied spatially with > 60% of the nuclei within active fascicles being positive. We interpret this high percentage to mean that many of the nuclei of preexisting muscle fibers must be expressing qmf1. The expression of the protooncogene c-myc (presumably by proliferating cells such as satellite cells, fibroblasts, and capillary epithelial cells) and the MRF qmf1 (by myoblasts and adult muscle nuclei) are among the early molecular responses of injured muscle. We conclude that myogenic regulatory factors are not permanently repressed after embryonic development and that derepression plays a role in the repair of terminally differentiated myofibers.
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Hardy, S., Y. Kong, and S. F. Konieczny. "Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain." Molecular and Cellular Biology 13, no. 10 (October 1993): 5943–56. http://dx.doi.org/10.1128/mcb.13.10.5943.
Full textAbstract:
MRF4 is a member of the muscle-specific basic helix-loop-helix transcription factor family that also includes MyoD, myogenin, and Myf-5. Each of these proteins, when overexpressed in fibroblasts, converts the cells to differentiated muscle fibers that express several skeletal muscle genes, such as those for alpha-actin, muscle creatine kinase, and troponin I. Despite the fact that MRF4 functions as a positive transcriptional regulator, the MRF4 protein is subject to negative regulation by a variety of agents, most notably by exposure of cells to purified growth factors, such as basic fibroblast growth factor (bFGF). In an effort to establish whether bFGF inhibits MRF4 activity through specific posttranslational modifications, we examined whether MRF4 exists in vivo as a phosphoprotein and whether the phosphorylation status of the protein regulates its activity. Our results indicate that MRF4 is phosphorylated predominantly on serine residues, with weak phosphorylation occurring on threonine residues. Both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylate MRF4 in vitro as well as in vivo, and the overexpression of each kinase inhibits MRF4 activity and thus blocks terminal differentiation. PKC-directed phosphorylation of a conserved threonine residue (T-99) situated within the DNA-binding domain inhibits MRF4 from binding in vitro to specific DNA targets. However, although T-99 itself is essential for myogenic activity, our studies demonstrate that the phosphorylation status of T-99 does not play a major role in regulating MRF4 activity in vivo, since PKA, PKC, and bFGF inhibit the activity of MRF4 proteins in which the identified PKA and PKC sites have been mutated. We suggest that the negative regulation of MRF4 imposed by bFGF does not involve a direct modification of the protein at the identified PKA and PKC sites but instead may involve the modification of specific coregulators that interact with this muscle regulatory factor.
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Hardy, S., Y. Kong, and S. F. Konieczny. "Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain." Molecular and Cellular Biology 13, no. 10 (October 1993): 5943–56. http://dx.doi.org/10.1128/mcb.13.10.5943-5956.1993.
Full textAbstract:
MRF4 is a member of the muscle-specific basic helix-loop-helix transcription factor family that also includes MyoD, myogenin, and Myf-5. Each of these proteins, when overexpressed in fibroblasts, converts the cells to differentiated muscle fibers that express several skeletal muscle genes, such as those for alpha-actin, muscle creatine kinase, and troponin I. Despite the fact that MRF4 functions as a positive transcriptional regulator, the MRF4 protein is subject to negative regulation by a variety of agents, most notably by exposure of cells to purified growth factors, such as basic fibroblast growth factor (bFGF). In an effort to establish whether bFGF inhibits MRF4 activity through specific posttranslational modifications, we examined whether MRF4 exists in vivo as a phosphoprotein and whether the phosphorylation status of the protein regulates its activity. Our results indicate that MRF4 is phosphorylated predominantly on serine residues, with weak phosphorylation occurring on threonine residues. Both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylate MRF4 in vitro as well as in vivo, and the overexpression of each kinase inhibits MRF4 activity and thus blocks terminal differentiation. PKC-directed phosphorylation of a conserved threonine residue (T-99) situated within the DNA-binding domain inhibits MRF4 from binding in vitro to specific DNA targets. However, although T-99 itself is essential for myogenic activity, our studies demonstrate that the phosphorylation status of T-99 does not play a major role in regulating MRF4 activity in vivo, since PKA, PKC, and bFGF inhibit the activity of MRF4 proteins in which the identified PKA and PKC sites have been mutated. We suggest that the negative regulation of MRF4 imposed by bFGF does not involve a direct modification of the protein at the identified PKA and PKC sites but instead may involve the modification of specific coregulators that interact with this muscle regulatory factor.
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Amacher, S. L., J. N. Buskin, and S. D. Hauschka. "Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle." Molecular and Cellular Biology 13, no. 5 (May 1993): 2753–64. http://dx.doi.org/10.1128/mcb.13.5.2753.
Full textAbstract:
We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.
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Amacher, S. L., J. N. Buskin, and S. D. Hauschka. "Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle." Molecular and Cellular Biology 13, no. 5 (May 1993): 2753–64. http://dx.doi.org/10.1128/mcb.13.5.2753-2764.1993.
Full textAbstract:
We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.
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Yang, Zhongyue, Md Shamimul Hasan, John K. Htoo, Derris D. Burnett, Jean M. Feugang, Mark A. Crenshaw, and Shengfa F. Liao. "Effects of dietary supplementation of l-methionine vs. dl-methionine on performance, plasma concentrations of free amino acids and other metabolites, and myogenesis gene expression in young growing pigs." Translational Animal Science 3, no. 1 (September 27, 2018): 329–39. http://dx.doi.org/10.1093/tas/txy109.
Full textAbstract:
Abstract Methionine (Met), the second or third limiting amino acid (AA) in typical swine diets, plays important roles in promoting swine health and growth, especially, muscle growth. Whereas dl-Met products have been used in swine industry for many years, l-Met products have been developed recently. This research was conducted to study the effects of supplemental l-Met or dl-Met on nutrient metabolism, muscle gene expression, and growth performance of pigs. Twenty crossbred young barrows (initial body weight [BW] 21.2 ± 2.7 kg) were randomly assigned to 20 individual pens and two dietary treatments according to a completely randomized design with pigs serving as the experiment unit (n = 10). Two corn and soybean meal-based diets (diets 1 and 2) were formulated to meet or exceed the recommended requirements for energy, AA, and other nutrients (NRC. 2012. Nutrient requirements of swine, 11th ed. Washington, DC: The National Academies Press; AMINODat 5.0). Crystalline l-Met and dl-Met were supplemented to diets 1 and 2 (both at 0.13%, as-fed basis), respectively. After 4 wk of an ad libitum feeding trial, BW and feed intake were measured to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed ratio (G:F). Blood samples were collected from the jugular vein for analyses of plasma AA and metabolite concentrations. The longissimus dorsi muscle samples were collected for analysis of myogenesis gene expression. Data were analyzed using Student’s t-test. There were no differences (P = 0.56 to 0.94) in ADG, ADFI, or G:F between pigs fed the two experimental diets and no differences between diets were observed in plasma free AA concentrations. No differences were observed between pigs fed the two diets in expression of mRNA for eight myogenesis-related genes, which were myogenic differentiation 1, myogenin, myogenic factors 5, muscle regulatory factor 4 (a.k.a. myogenic factors 6), and myocyte enhancer factors 2A, 2B, 2C, and 2D. In conclusion, results of this experiment indicate that the bioefficacy of l-Met is not different from that of dl-Met, which is likely because of an efficient conversion of d-Met to l-Met by pigs.
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Quigley, S. P., P. L. Greenwood, D. O. Kleemann, J. A. Owens, C. S. Bawden, and G. S. Nattrass. "Myogenesis in small and large ovine fetuses at three stages of pregnancy." Animal Production Science 55, no. 2 (2015): 207. http://dx.doi.org/10.1071/an14203.
Full textAbstract:
Perturbations of the prenatal environment may influence fetal muscle development. This study investigated muscle cellularity and mRNA abundance of myogenic genes in fetal sheep divergent in their patterns of growth. Muscle samples were obtained from small and large fetuses on Days 50, 92 and 133 of pregnancy. Number of myofibres in the semitendinosus muscle increased between Day 92 and 133 of pregnancy, but did not differ between small and large fetuses at either stage of pregnancy. The semitendinosus of small fetuses had smaller cross-sectional areas of myofibres than did those of their large counterparts on Day 133 of pregnancy. The semitendinosus of small fetuses also had lower DNA concentration on Day 92 and lower protein concentration on Day 133 than did those of large fetuses. The mRNA levels of the myogenic regulatory factors (MRFs), myostatin, the insulin-like growth factors and embryonic myosin in fetal muscles varied with the stage of development, but no differences occurred in response to divergent fetal growth. Myostatin mRNA was more abundant in the semitendinosus than in the supraspinatus muscle on Days 92 and 133, as were myogenic regulatory factors, myf-5, myf-6 and follistatin mRNA on Day 133. The results indicated that muscle growth but not the number of myofibres in fetal sheep is modified by restricted fetal growth, and that genes that regulate muscle development are affected by the stage of development in an anatomical muscle-specific manner.
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Christensen, T. H., H. Prentice, R. Gahlmann, and L. Kedes. "Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements." Molecular and Cellular Biology 13, no. 11 (November 1993): 6752–65. http://dx.doi.org/10.1128/mcb.13.11.6752.
Full textAbstract:
The cardiac troponin C (cTnC) gene produces identical transcripts in slow-twitch skeletal muscle and in heart muscle (R. Gahlmann, R. Wade, P. Gunning, and L. Kedes, J. Mol. Biol. 201:379-391, 1988). A separate gene encodes the fast-twitch skeletal muscle troponin C and is not expressed in heart muscle. We have used transient transfection to characterize the regulatory elements responsible for skeletal and cardiac cell-type-specific expression of the human cTnC (HcTnC) gene. At least four separate elements cooperate to confer tissue-specific expression of this gene in differentiated myotubes; a basal promoter (between -61 and -13) augments transcription 9-fold, upstream major regulatory sequences (between -68 and -142 and between -1319 and -4500) augment transcription as much as 39-fold, and at least two enhancer-like elements in the first intron (between +58 and +1028 and between +1029 and +1523) independently augment transcription 4- to 5-fold. These enhancers in the first intron increase myotube-specific chloramphenicol acetyltransferase activity when linked to their own promoter elements or to the heterologous simian virus 40 promoter, and the effects are multiplicative rather than additive. Each of the major myotube regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD.A MyoD expression vector in 10T1/2 cells induced constructs carrying either the upstream HcTnC promoter elements or the first intron of the gene 300- to 500-fold. Expression was inhibited by cotransfection with Id, a negative regulator of basic helix-loop-helix transcription factors. The basal promoter contains five tandem TGGGC repeats that interact with Sp1 or an Sp1-like factor in nuclear extracts. Mutational analysis of this element demonstrated that two of the five repeat sequences were sufficient to support basal level muscle cell-specific transcription. Whereas the basal promoter is also critical for expression in cardiac myocytes, the elements upstream of -67 appear to play little or no role. Major augmentation of expression in cardiomyocytes is also provided by sequences in the first intron, but these are upstream (between +58 and +1028). The downstream segment of the first intron has no enhancer activity in cardiomyocytes. A specific DNA-protein complex is formed by this C2 cell enhancer with extracts from C2 cells but not cardiomyocytes. These observations suggest that tissue-specific expression of the HcTnC gene is cooperatively regulated by the complex interactions of multiple regulatory elements and that different elements are used to regulate expression in myogenic and cardiac cells.
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Christensen, T. H., H. Prentice, R. Gahlmann, and L. Kedes. "Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements." Molecular and Cellular Biology 13, no. 11 (November 1993): 6752–65. http://dx.doi.org/10.1128/mcb.13.11.6752-6765.1993.
Full textAbstract:
The cardiac troponin C (cTnC) gene produces identical transcripts in slow-twitch skeletal muscle and in heart muscle (R. Gahlmann, R. Wade, P. Gunning, and L. Kedes, J. Mol. Biol. 201:379-391, 1988). A separate gene encodes the fast-twitch skeletal muscle troponin C and is not expressed in heart muscle. We have used transient transfection to characterize the regulatory elements responsible for skeletal and cardiac cell-type-specific expression of the human cTnC (HcTnC) gene. At least four separate elements cooperate to confer tissue-specific expression of this gene in differentiated myotubes; a basal promoter (between -61 and -13) augments transcription 9-fold, upstream major regulatory sequences (between -68 and -142 and between -1319 and -4500) augment transcription as much as 39-fold, and at least two enhancer-like elements in the first intron (between +58 and +1028 and between +1029 and +1523) independently augment transcription 4- to 5-fold. These enhancers in the first intron increase myotube-specific chloramphenicol acetyltransferase activity when linked to their own promoter elements or to the heterologous simian virus 40 promoter, and the effects are multiplicative rather than additive. Each of the major myotube regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD.A MyoD expression vector in 10T1/2 cells induced constructs carrying either the upstream HcTnC promoter elements or the first intron of the gene 300- to 500-fold. Expression was inhibited by cotransfection with Id, a negative regulator of basic helix-loop-helix transcription factors. The basal promoter contains five tandem TGGGC repeats that interact with Sp1 or an Sp1-like factor in nuclear extracts. Mutational analysis of this element demonstrated that two of the five repeat sequences were sufficient to support basal level muscle cell-specific transcription. Whereas the basal promoter is also critical for expression in cardiac myocytes, the elements upstream of -67 appear to play little or no role. Major augmentation of expression in cardiomyocytes is also provided by sequences in the first intron, but these are upstream (between +58 and +1028). The downstream segment of the first intron has no enhancer activity in cardiomyocytes. A specific DNA-protein complex is formed by this C2 cell enhancer with extracts from C2 cells but not cardiomyocytes. These observations suggest that tissue-specific expression of the HcTnC gene is cooperatively regulated by the complex interactions of multiple regulatory elements and that different elements are used to regulate expression in myogenic and cardiac cells.
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Temple, Genevieve K., Nicholas J. Cole, and Ian A. Johnston. "Embryonic temperature and the relative timing of muscle-specific genes during development in herring (Clupea harengus L.)." Journal of Experimental Biology 204, no. 21 (November 1, 2001): 3629–37. http://dx.doi.org/10.1242/jeb.204.21.3629.
Full textAbstract:
SUMMARY Temperature influences many aspects of muscle development in herring (Clupea harengus). In Clyde herring, myofibril synthesis occurred later with respect to somite stage in embryos reared at 5°C compared with 12°C. The aim of the present study was to test the hypothesis that the relative timing of expression of myogenic regulatory factors (MRFs) and myosin heavy chain (MyHC) transcripts changes with developmental temperature. Reverse transcriptase/polymerase chain reaction (RT-PCR) was used to clone partial coding regions of MyoD, myogenin and MyHC from juvenile Clyde herring. Embryos were reared at 5, 8 and 12°C, and the spatial and temporal expression patterns of transcripts were investigated using cRNA probes and in situ hybridisation. Antisense probes revealed a rostral–caudal progression of all three transcripts. MyoD transcription initially took place in the adaxial cells of the unsegmented, presomitic mesoderm, whereas myogenin transcription first occurred in newly formed somites. The MyHC gene transcript was not detected until approximately nine somites had formed. Since the somite stage at which the MRFs and MyHC were first expressed was independent of temperature, the hypothesis was rejected. We suggest that the effects of temperature on myofibril synthesis must occur downstream from MyHC transcription either at the level of translation or at the assembly stage.
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van der Velden, Jos L. J., Ramon C. J. Langen, Marco C. J. M. Kelders, Emiel F. M. Wouters, Yvonne M. W. Janssen-Heininger, and Annemie M. W. J. Schols. "Inhibition of glycogen synthase kinase-3β activity is sufficient to stimulate myogenic differentiation." American Journal of Physiology-Cell Physiology 290, no. 2 (February 2006): C453—C462. http://dx.doi.org/10.1152/ajpcell.00068.2005.
Full textAbstract:
Skeletal muscle atrophy is a prominent and disabling feature of chronic wasting diseases. Prevention or reversal of muscle atrophy by administration of skeletal muscle growth (hypertrophy)-stimulating agents such as insulin-like growth factor I (IGF-I) could be an important therapeutic strategy in these diseases. To elucidate the IGF-I signal transduction responsible for muscle formation (myogenesis) during muscle growth and regeneration, we applied IGF-I to differentiating C2C12 myoblasts and evaluated the effects on phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK-3β) signaling and myogenesis. IGF-I caused phosphorylation and inactivation of GSK-3β activity via signaling through the PI3K/Akt pathway. We assessed whether pharmacological inhibition of GSK-3β with lithium chloride (LiCl) was sufficient to stimulate myogenesis. Addition of IGF-I or LiCl stimulated myogenesis, evidenced by increased myotube formation, muscle creatine kinase (MCK) activity, and troponin I (TnI) promoter transactivation during differentiation. Moreover, mRNAs encoding MyoD, Myf-5, myogenin, TnI-slow, TnI-fast, MCK, and myoglobin were upregulated in myoblasts differentiated in the presence of IGF-I or LiCl. Importantly, blockade of GSK-3β inhibition abrogated IGF-I- but not LiCl-dependent stimulation of myogenic mRNA accumulation, suggesting that the promyogenic effects of IGF-I require GSK-3β inactivation and revealing an important negative regulatory role for GSK-3β in myogenesis. Therefore, this study identifies GSK-3β as a potential target for pharmacological stimulation of muscle growth.
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Hawke, Thomas J., Shane B. Kanatous, Cindy M. Martin, Sean C. Goetsch, and Daniel J. Garry. "Rad is temporally regulated within myogenic progenitor cells during skeletal muscle regeneration." American Journal of Physiology-Cell Physiology 290, no. 2 (February 2006): C379—C387. http://dx.doi.org/10.1152/ajpcell.00270.2005.
Full textAbstract:
The successful use of myogenic progenitor cells for therapeutic applications requires an understanding of the intrinsic and extrinsic cues involved in their regulation. Herein we demonstrate the expression pattern and transcriptional regulation of Rad, a prototypical member of a family of novel Ras-related GTPases, during mammalian development and skeletal muscle regeneration. Rad was identified using microarray analysis, which revealed robust upregulation of its expression during skeletal muscle regeneration. Our current findings demonstrate negligible Rad expression with resting adult skeletal muscle; however, after muscle injury, Rad is expressed within the myogenic progenitor cell population. Rad expression is significantly increased and localized to the myogenic progenitor cell population during the early phases of regeneration and within the newly regenerated myofibers during the later phases of regeneration. Immunohistochemical analysis demonstrated that Rad and MyoD are coexpressed within the myogenic progenitor cell population of regenerating skeletal muscle. This expression profile of Rad during skeletal muscle regeneration is consistent with the proposed roles for Rad in the inhibition of L-type Ca2+channel activity and the inhibition of Rho/RhoA kinase activity. We also have demonstrated that known myogenic transcription factors (MEF2, MyoD, and Myf-5) can increase the transcriptional activity of the Rad promoter and that this ability is significantly enhanced by the presence of the Ca2+-dependent phosphatase calcineurin. Furthermore, this enhanced transcriptional activity appears to be dependent on the presence of a conserved NFAT binding motif within the Rad promoter. Taken together, these data define Rad as a novel factor within the myogenic progenitor cells of skeletal muscle and identify key regulators of its transcriptional activity.
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Parmacek, M. S., H. S. Ip, F. Jung, T. Shen, J. F. Martin, A. J. Vora, E. N. Olson, and J. M. Leiden. "A novel myogenic regulatory circuit controls slow/cardiac troponin C gene transcription in skeletal muscle." Molecular and Cellular Biology 14, no. 3 (March 1994): 1870–85. http://dx.doi.org/10.1128/mcb.14.3.1870.
Full textAbstract:
The slow/cardiac troponin C (cTnC) gene is expressed in three distinct striated muscle lineages: cardiac myocytes, embryonic fast skeletal myotubes, and adult slow skeletal myocytes. We have reported previously that cTnC gene expression in cardiac muscle is regulated by a cardiac-specific promoter/enhancer located in the 5' flanking region of the gene (bp -124 to +1). In this report, we demonstrate that the cTnC gene contains a second distinct and independent transcriptional enhancer which is located in the first intron. This second enhancer is skeletal myotube specific and is developmentally up-regulated during the differentiation of myoblasts to myotubes. This enhancer contains three functionally important nuclear protein binding sites: a CACCC box, a MEF-2 binding site, and a previously undescribed nuclear protein binding site, designated MEF-3, which is also present in a large number of skeletal muscle-specific transcriptional enhancers. Unlike most skeletal muscle-specific transcriptional regulatory elements, the cTnC enhancer does not contain a consensus binding site (CANNTG) for the basic helix-loop-helix (bHLH) family of transcription factors and does not directly bind MyoD-E12 protein complexes. Despite these findings, the cTnC enhancer can be transactivated by overexpression of the myogenic bHLH proteins, MyoD and myogenin, in C3H10T1/2 (10T1/2) cells. Electrophoretic mobility shift assays demonstrated changes in the patterns of MEF-2, CACCC, and MEF-3 DNA binding activities following the conversion of 10T1/2 cells into myoblasts and myotubes by stable transfection with a MyoD expression vector. In particular, MEF-2 binding activity was up-regulated in 10T1/2 cells stably transfected with a MyoD expression vector only after these cells fused and differentiated into skeletal myotubes. Taken together, these results demonstrated that distinct lineage-specific transcriptional regulatory elements control the expression of a single myofibrillar protein gene in fast skeletal and cardiac muscle. In addition, they show that bHLH transcription factors can indirectly transactivate the expression of some muscle-specific genes.