Molecular and Cellular Biochemistry

, Volume 344, Issue 1–2, pp 151–162 | Cite as

Expression of myocyte enhancer factor-2 and downstream genes in ground squirrel skeletal muscle during hibernation

  • Shannon N. Tessier
  • Kenneth B. Storey


Myocyte enhancer factor-2 (MEF2) transcription factors regulate the expression of a variety of genes encoding contractile proteins and other proteins associated with muscle performance. We proposed that changes in MEF2 levels and expression of selected downstream targets would aid the skeletal muscle of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) in meeting metabolic challenges associated with winter hibernation; e.g., cycles of torpor–arousal, body temperature that can fall to near 0°C, long periods of inactivity that could lead to atrophy. MEF2A protein levels were significantly elevated when animals were in torpor (maximally 2.8-fold higher than in active squirrels) and the amount of phosphorylated active MEF2A Thr312 increased during entrance into torpor. MEF2C levels also rose significantly during entrance and torpor as did the amount of phosphorylated MEF2C Ser387. Furthermore, both MEF2 members showed elevated amounts in the nuclear fraction during torpor as well as enhanced binding to DNA indicating that MEF2-mediated gene expression was up-regulated in torpid animals. Indeed, the protein products of two MEF2 downstream gene targets increased in muscle during torpor (glucose transporter isoforms 4; GLUT4) or early arousal (myogenic differentiation; MyoD). Significant increases in Glut4 and MyoD mRNA transcript levels correlated with the rise in protein product levels and provided further support for the activation of MEF2-mediated gene expression in the hibernator. Transcript levels of Mef2a and Mef2c also showed time-dependent patterns with levels of both being highest during arousal from torpor. The data suggest a significant role for MEF2-mediated gene transcription in the selective adjustment of muscle protein complement over the course of torpor–arousal cycles.


Spermophilustridecemlineatus Hibernation Muscle atrophy Myocyte enhancer factor-2 Western blots RT-PCR 



We thank Dr. J.M. Hallenbeck (National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD) for providing the tissue samples for this study. Thank also to B. Lant and J.M. Storey for editorial review of the manuscript. Research was supported by a discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to KBS and the Canada Research Chairs program. SNT held a NSERC PGSM scholarship.


  1. 1.
    Staples JF, Brown JC (2008) Mitochondrial metabolism in hibernation and daily torpor: a review. J Comp Physiol B 178:811–827CrossRefPubMedGoogle Scholar
  2. 2.
    Wang LCH, Lee TF (1996) Torpor and hibernation in mammals: metabolic, physiological, and biochemical adaptations. In: Fregley MJ, Blatteis CM (eds) Handbook of physiology: environmental physiology sect 4, vol 1. Oxford University Press, New York, pp 507–532Google Scholar
  3. 3.
    Storey KB (2010) Out cold: biochemical regulation of mammalian hibernation—a mini-review. Gerontology 56(2):220–230CrossRefPubMedGoogle Scholar
  4. 4.
    Geiser F (2004) Metabolic rate and body temperature reduction during hibernation and daily torpor. Annu Rev Physiol 66:239–274CrossRefPubMedGoogle Scholar
  5. 5.
    Carey HV, Andrews MT, Martin SL (2003) Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperatures. Physiol Rev 83:1153–1181PubMedGoogle Scholar
  6. 6.
    Nelson CJ, Otis JP, Martin SL, Carey HV (2009) Analysis of the hibernation cycle using LC-MS based metabolomics in ground squirrel liver. Physiol Genomics 37:43–51CrossRefPubMedGoogle Scholar
  7. 7.
    Fahlman A, Storey JM, Storey KB (2000) Gene up-regulation in heart during mammalian hibernation. Cryobiology 40:332–342CrossRefPubMedGoogle Scholar
  8. 8.
    Morin PJ, Ni Z, McMullen DC, Storey KB (2008) Expression of Nrf2 and its downstream gene targets in hibernating 13-lined ground squirrels, Spermophilus tridecemlineatus. Mol Cell Biochem 312:121–129CrossRefPubMedGoogle Scholar
  9. 9.
    Morin PJ, Storey KB (2007) Antioxidant defense in hibernation: cloning and expression of peroxiredoxins from hibernating ground squirrels, Spermophilus tridecemlineatus. Arch Biochem Biophys 461:59–65CrossRefPubMedGoogle Scholar
  10. 10.
    Morin PJ, Storey KB (2006) Evidence of a reduced transcriptional state during hibernation in ground squirrels. Cryobiology 53:310–318CrossRefPubMedGoogle Scholar
  11. 11.
    Mamady H, Storey KB (2006) Up-regulation of the endoplasmic reticulum molecular chaperone GRP78 during hibernation in thirteen-lined ground squirrels. Mol Cell Biochem 292:89–98CrossRefPubMedGoogle Scholar
  12. 12.
    Joanisse DR (2004) Skeletal muscle metabolism and plasticity. Functional metabolism: regulation and adaptation. Wiley, New YorkGoogle Scholar
  13. 13.
    Malatesta M, Perdoni F, Battistelli S, Muller S, Zancanaro C (2009) The cell nuclei of skeletal muscle cells are transcriptionally active in hibernating edible dormice. MBC Cell Biol 10:19Google Scholar
  14. 14.
    Choi H, Selpides PJ, Nowell MM, Rourke BC (2009) Functional overload in ground squirrels plantaris muscle fails to induce myosin isoform shifts. Am J Physiol Regul Integr Comp Physiol 297(3):R578–R586PubMedGoogle Scholar
  15. 15.
    Bassel-Duby R, Olson EN (2006) Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 75:19–37CrossRefPubMedGoogle Scholar
  16. 16.
    Rourke BC, Yokoyama Y, Milsom WK, Caiozzo VJ (2004) Myosin isoform expression and MAFbx mRNA levels in hibernating golden mantled ground squirrels (Spermophilus lateralis). Physiol Biochem Zool 77:582–593CrossRefPubMedGoogle Scholar
  17. 17.
    Shavlakadze T, Grounds M (2006) Of bears, frogs, meat, mice and men: complexity of factors affecting skeletal muscle mass and fat. Bioessays 28:994–1009CrossRefPubMedGoogle Scholar
  18. 18.
    Yacoe M (1983) Maintenance of the pectoralis muscle during hibernation in the big brown bat Eptesicus fuscus. J Comp Physiol 152:137–144Google Scholar
  19. 19.
    Lee K, Park JY, Yoo W, Gwag T, Lee JW, Byun MW, Choi I (2008) Overcoming muscle atrophy in a hibernating mammal despite prolonged disuse in dormancy: proteomic and molecular assessment. J Cell Biochem 104:642–656CrossRefPubMedGoogle Scholar
  20. 20.
    Rourke BC, Cotton CJ, Harlow HJ, Caiozzo VJ (2006) Maintenance of slow type I myosin protein and mRNA expression in overwintering prairie dogs and black bears. J Comp Physiol B 176:709–720CrossRefPubMedGoogle Scholar
  21. 21.
    Musacchia XJ, Steffen JM, Steffen MC, Geoghegan TE, Dombrowski JM, Milsom WK, Burlington RF (1989) Morphometric and biochemical adaptations of skeletal muscle in hibernating and non-hibernating ground squirrels, S. lateralis. In: Malan A, Canguilhem B (eds) Living in the cold. INSERM, Paris, pp 217–224Google Scholar
  22. 22.
    Wickler SJ, Hoyt DF, van Breukelen F (1991) Disuse atrophy in the hibernating golden-mantled ground squirrel, Spermophilus lateralis. Am J Physiol 261(5 Pt 2):R1214–R1217PubMedGoogle Scholar
  23. 23.
    Storey KB, Storey JM (2010) Metabolic rate depression: the biochemistry of mammalian hibernation. Clin Chem (in press)Google Scholar
  24. 24.
    Caiozzo VJ (2002) Plasticity of skeletal muscle phenotype: mechanical consequences. Muscle Nerve 26:740–768CrossRefPubMedGoogle Scholar
  25. 25.
    Zuikova OV, Osipova DA, Vikhliantsev IM, Malyshev SL, Udal’tsov SL, Podlubnaia ZA (2005) Myosin light chains of skeletal and cardiac muscles of ground squirrels Citellus undulatus in different periods of hibernation. Biofizika 50(5):797–802PubMedGoogle Scholar
  26. 26.
    Mamady H, Storey KB (2008) Coping with stress: expression of ATF4, ATF6 and downstream targets in organs of hibernating ground squirrels. Arch Biochem Biophys 477(1):77–85CrossRefPubMedGoogle Scholar
  27. 27.
    Ni Z, Storey KB (2010) Heme oxygenase expression and Nrf2 signaling during hibernation in ground squirrels. Can J Physiol Pharmacol 88(3):379–387CrossRefPubMedGoogle Scholar
  28. 28.
    Black BL, Olson EN (1998) Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu Rev Cell Dev Biol 14:167–196CrossRefPubMedGoogle Scholar
  29. 29.
    Kim MS, Fielitz J, McAnnally J, Shelton JM, Lemon DD, McKinsey TA, Richardson JA, Bassel-Duby R, Olson EN (2008) Protein kinase D1 stimulates MEF2 activity in skeletal muscle and enhances muscle performance. Mol Cell Biol 28:3600–3609CrossRefPubMedGoogle Scholar
  30. 30.
    McMullen DC, Hallenbeck JM (2010) Regulation of Akt during torpor in the hibernating ground squirrel, Ictidomys tridecemlineatus. J Comp Physiol B. doi: 10.1007/s00360-010-0468-8
  31. 31.
    Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489CrossRefPubMedGoogle Scholar
  32. 32.
    Harlow HJ, Lohuis T, Beck TD, Iaizzo PA (2001) Muscle strength in overwintering bears. Nature 409(6823):997CrossRefPubMedGoogle Scholar
  33. 33.
    Tinker DB, Harlow HJ, Beck TD (1998) Protein use and muscle-fiber changes in free-ranging, hibernating black bears. Physiol Zool 71:414–424CrossRefPubMedGoogle Scholar
  34. 34.
    Cotton CJ, Harlow HJ (2010) Avoidance of skeletal muscle atrophy in spontaneous and facultative hibernators. Physiol Biochem Zool 83(3):551–560CrossRefPubMedGoogle Scholar
  35. 35.
    Ishikawa F, Miyoshi H, Nose K, Shibanuma M (2010) Transcriptional induction of MMP-10 by TGF-beta, mediated by activation of MEF2A and downregulation of class IIa HDACs. Oncogene 29(6):909–919CrossRefPubMedGoogle Scholar
  36. 36.
    Sun W, Wei X, Kesavan K, Garrington TP, Fan R, Mei J, Anderson SM, Gelfand EW, Johnson GL (2003) MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src. Mol Cell Biol 23(7):2298–2308CrossRefPubMedGoogle Scholar
  37. 37.
    Chandran R, Knobloch TJ, Anghelina M, Agarwal S (2007) Biomechanical signals upregulate myogenic gene induction in the presence or absence of inflammation. Am J Physiol Cell Physiol 293(1):C267–C276CrossRefPubMedGoogle Scholar
  38. 38.
    Olson EN (2004) Undermining the endothelium by ablation of MAPK-MEF2 signaling. J Clin Invest 113:1110–1112PubMedGoogle Scholar
  39. 39.
    Cox DM, Du M, Marback M, Yang EC, Chan J, Siu KW, McDermott JC (2003) Phosphorylation motifs regulating the stability and function of myocyte enhancer factor 2A. J Biol Chem 278:15297–15303CrossRefPubMedGoogle Scholar
  40. 40.
    Rampalli S, Li L, Mak E, Ge K, Brand M, Tapscott SJ, Dilworth FJ (2007) p38 MAPK signaling regulates recruitment of Ash2L-containing methyltransferase complexes to specific genes during differentiation. Nat Struct Mol Biol 14:1150–1156CrossRefPubMedGoogle Scholar
  41. 41.
    Ma K, Chan JK, Zhu G, Wu Z (2005) Myocyte enhancer factor 2 acetylation by p300 enhances its DNA binding activity, transcriptional activity, and myogenic differentiation. Mol Cell Biol 25:3575–3582CrossRefPubMedGoogle Scholar
  42. 42.
    Sun W, Wei X, Kesavan K, Garrington TP, Fan R, Mei J, Anderson SM, Gelfand EW, Johnson GL (2003) MEK kinase 2 and the adaptor protein lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src. Mol Cell Biol 23:2298–2308CrossRefPubMedGoogle Scholar
  43. 43.
    Kato Y, Kravchenko VV, Tapping RI, Han J, Ulevitch RJ, Lee JD (1997) BMK1/ERK5 regulates serum-induced early gene expression through transcription factor MEF2C. EMBO J 16:7054–7066CrossRefPubMedGoogle Scholar
  44. 44.
    Eddy SF, Storey KB (2007) p38 MAPK regulation of transcription factor targets in muscle and heart of the hibernating bat, Myotis lucifugus. Cell Biochem Funct 25:759–765CrossRefPubMedGoogle Scholar
  45. 45.
    MacDonald JA, Storey KB (2005) Mitogen-activated protein kinases and selected downstream targets display organ-specific responses in the hibernating ground squirrel. Int J Biochem Cell Biol 37:679–691CrossRefPubMedGoogle Scholar
  46. 46.
    McKinsey TA, Zhang CL, Olson EN (2000) Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14–3-3 to histone deacetylase 5. Proc Natl Acad Sci USA 97:14400–14405CrossRefPubMedGoogle Scholar
  47. 47.
    Yoon SC, Ahn YM, Jun SJ, Kim Y, Kang UG, Park JB, Kim YS (2005) Region-specific phosphorylation of ERK5-MEF2C in the rat frontal cortex and hippocampus after electroconvulsive shock. Prog Neuropsychopharmacol Biol Psychiatry 29:749–753CrossRefPubMedGoogle Scholar
  48. 48.
    Berkes CA, Tapscott SJ (2005) MyoD and the transcriptional control of myogenesis. Semin Cell Dev Biol 16:585–595CrossRefPubMedGoogle Scholar
  49. 49.
    Naya FJ, Olson E (1999) MEF2: a transcriptional target for signaling pathways controlling skeletal muscle growth and differentiation. Curr Opin Cell Biol 11:683–688CrossRefPubMedGoogle Scholar
  50. 50.
    Legerlotz K, Smith HK (2008) Role of MyoD in denervated, disused, and exercised muscle. Muscle Nerve 38:1087–1100CrossRefPubMedGoogle Scholar
  51. 51.
    De Falco G, Comes F, Simone C (2006) pRb: master of differentiation. Coupling irreversible cell cycle withdrawal with induction of muscle-specific transcription. Oncogene 25:5244–5249CrossRefPubMedGoogle Scholar
  52. 52.
    Wright DC (2007) Mechanisms of calcium-induced mitochondrial biogenesis and GLUT4 synthesis. Appl Physiol Nutr Metab 32:840–845CrossRefPubMedGoogle Scholar
  53. 53.
    McGee SL, Hargreaves M (2006) Exercise and skeletal muscle glucose transporter 4 expression: molecular mechanisms. Clin Exp Pharmacol Physiol 33:395–399CrossRefPubMedGoogle Scholar
  54. 54.
    Holloszy JO (2008) Regulation by exercise of skeletal muscle content of mitochondria and GLUT4. J Physiol Pharmacol 59(Suppl 7):5–18PubMedGoogle Scholar
  55. 55.
    Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O, Michael LF, Puiqserver P, Isotani E, Olson EN, Lowell BB, Bassel-Duby R, Spiegelman BM (2002) Transcriptional co-activator PCG-1 alpha drives the formation of slow-twitch muscle fibers. Nature 418(6899):797–801CrossRefPubMedGoogle Scholar
  56. 56.
    Eddy SF, Morin PJ, Storey KB (2005) Cloning and expression of PPARγ and PGC-1α from the hibernating ground squirrel, Spermophilus tridecemlineatus. Mol Cell Biochem 269:175–182CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  1. 1.Institute of Biochemistry & Department of BiologyCarleton UniversityOttawaCanada

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