Biotechnology Letters

, Volume 36, Issue 5, pp 1113–1124 | Cite as

Acute mechanical overload increases IGF-I and MMP-9 mRNA in 3D tissue-engineered skeletal muscle

  • D. J. Player
  • N. R. W. Martin
  • S. L. Passey
  • A. P. Sharples
  • V. Mudera
  • M. P. LewisEmail author
Original Research Paper


Skeletal muscle (SkM) is a tissue that responds to mechanical load following both physiological (exercise) or pathophysiological (bed rest) conditions. The heterogeneity of human samples and the experimental and ethical limitations of animal studies provide a rationale for the study of SkM plasticity in vitro. Many current in vitro approaches of mechanical loading of SkM disregard the three-dimensional (3D) structure in vivo. Tissue engineered 3D SkM, that displays highly aligned and differentiated myotubes, was used to investigate mechano-regulated gene transcription of genes implicated in hypertrophy/atrophy. Static loading (STL) and ramp loading (RPL) at 10 % strain for 60 min were used as mechano-stimulation with constructs sampled immediately for RNA extraction. STL increased IGF-I mRNA compared to both RPL and CON (control, p = 0.003 and 0.011 respectively) whilst MMP-9 mRNA increased in STL and RPL compared to CON (both p < 0.05). IGFBP-2 mRNA was differentially regulated in RPL and STL compared to CON (p = 0.057), whilst a reduction in IGFBP-5 mRNA was found for STL and RPL compared to CON (both p < 0.05). There was no effect in the expression of putative atrophic genes, myostatin, MuRF-1 and MAFBx (all p > 0.05). These data demonstrate a transcriptional signature associated with SkM hypertrophy within a tissue-engineered model that more greatly recapitulates the in vivo SkM structure compared previously published studies.


Mechanical load Hypertrophy Myotubes Insulin-like growth factor binding proteins Tissue engineering 

Supplementary material

10529_2014_1464_MOESM1_ESM.docx (13 kb)
Supplementary material 1 (DOCX 13 kb)


  1. Adams GR, Haddad F (1996) The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy. J Appl Physiol 81:2509–2516PubMedGoogle Scholar
  2. Adams GR, McCue SA (1998) Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats. J Appl Physiol 84:1716–1722PubMedGoogle Scholar
  3. Adams GR, Haddad F, Baldwin KM (1999) Time course of changes in markers of myogenesis in overloaded rat skeletal muscles. J Appl Physiol 87:1705–1712PubMedGoogle Scholar
  4. Aguiar AF, Vechetti-Junior IJ, Alves de Souza RW, Castan EP, Milanezi-Aguiar RC, Padovani CR, Carvalho RF, Silva MD (2012) Myogenin, MyoD and IGF-I regulate muscle mass but not fiber-type conversion during resistance training in rats. Int J Sports Med 34:293–301PubMedCrossRefGoogle Scholar
  5. Armstrong RB, Marum P, Tullson P, Saubertt CW 4th (1979) Acute hypertrophic response of skeletal muscle to removal of synergists. J Appl Physiol Resp Environ Exerc Physiol 46:835–842Google Scholar
  6. Auluck A, Mudera V, Hunt NP, Lewis MP (2005) A three-dimensional in vitro model system to study the adaptation of craniofacial skeletal muscle following mechanostimulation. Eur J Oral Sci 113:218–224PubMedCrossRefGoogle Scholar
  7. Awede B, Thissen J, Gailly P, Lebacq J (1999) Regulation of IGF-I, IGFBP-4 and IGFBP-5 gene expression by loading in mouse skeletal muscle. FEBS Lett 461:263–267PubMedCrossRefGoogle Scholar
  8. Awede BL, Thissen JP, Lebacq J (2002) Role of IGF-I and IGFBPs in the changes of mass and phenotype induced in rat soleus muscle by clenbuterol. Am J Physiol Endocrinol Metab 282:E31–E37PubMedGoogle Scholar
  9. Bickel CS, Slade JM, Haddad F, Adams GR, Dudley GA (2003) Acute molecular responses of skeletal muscle to resistance exercise in able-bodied and spinal cord-injured subjects. J Appl Physiol 94:2255–2262PubMedGoogle Scholar
  10. Bickel CS, Slade J, Mahoney E, Haddad F, Dudley GA, Adams GR (2005) Time course of molecular responses of human skeletal muscle to acute bouts of resistance exercise. J Appl Physiol 98:482–488PubMedGoogle Scholar
  11. Blau HM, Pavlath GK, Hardeman EC, Chiu CP, Silberstein L, Webster SG, Miller SC, Webster C (1985) Plasticity of the differentiated state. Science 230:758–766PubMedCrossRefGoogle Scholar
  12. Carmeli E, Coleman R, Reznick AZ (2002) The biochemistry of aging muscle. Exp Gerontol 37:477–489PubMedCrossRefGoogle Scholar
  13. Caron MA, Charette SJ, Maltais F, Debigare R (2011) Variability of protein level and phosphorylation status caused by biopsy protocol design in human skeletal muscle analyses. BMC Res Notes 4:488PubMedCentralPubMedCrossRefGoogle Scholar
  14. Carson JA, Nettleton D, Reecy JM (2002) Differential gene expression in the rat soleus muscle during early work overload-induced hypertrophy. FASEB J 16:207–209PubMedGoogle Scholar
  15. Cheema U, Yang SY, Mudera V, Goldspink GG, Brown RA (2003) 3-D in vitro model of early skeletal muscle development. Cell Motil Cytoskelet 54:226–236CrossRefGoogle Scholar
  16. Cheema U, Brown R, Mudera V, Yang SY, McGrouther G, Goldspink G (2005) Mechanical signals and IGF-I gene splicing in vitro in relation to development of skeletal muscle. J Cell Physiol 202:67–75PubMedCrossRefGoogle Scholar
  17. Clemmons DR (1998) Role of insulin-like growth factor binding proteins in controlling IGF actions. Mol Cell Endocrinol 140:19–24PubMedCrossRefGoogle Scholar
  18. Coppock HA, White A, Aplin JD, Westwood M (2004) Matrix metalloprotease-3 and -9 proteolyze insulin-like growth factor-binding protein-1. Biol Reprod 71:438–443PubMedCrossRefGoogle Scholar
  19. Cuthbertson DJ, Babraj J, Smith K, Wilkes E, Fedele MJ, Esser K, Rennie M (2006) Anabolic signaling and protein synthesis in human skeletal muscle after dynamic shortening or lengthening exercise. Am J Physiol Endocrinol Metab 290:E731–E738PubMedCrossRefGoogle Scholar
  20. Dahiya S, Bhatnagar S, Hindi SM, Jiang C, Paul PK, Kuang S, Kumar A (2011) Elevated levels of active matrix metalloproteinase-9 cause hypertrophy in skeletal muscle of normal and dystrophin-deficient mdx mice. Hum Mol Genet 20:4345–4359PubMedCentralPubMedCrossRefGoogle Scholar
  21. Degens H, Alway SE (2006) Control of muscle size during disuse, disease, and aging. Int J Sports Med 27:94–99PubMedCrossRefGoogle Scholar
  22. Eastwood M, McGrouther DA, Brown RA (1998a) Fibroblast responses to mechanical forces. Proc Inst Mech Eng H 212:85–92PubMedCrossRefGoogle Scholar
  23. Eastwood M, Mudera VC, McGrouther DA, Brown RA (1998b) Effect of precise mechanical loading on fibroblast populated collagen lattices: morphological changes. Cell Motil Cytoskelet 40:13–21CrossRefGoogle Scholar
  24. Esser KA, White TP (1995) Mechanical load affects growth and maturation of skeletal muscle grafts. J Appl Physiol 78:30–37PubMedCrossRefGoogle Scholar
  25. Friedmann-Bette B, Schwartz FR, Eckhardt H, Billeter R, Bonaterra G, Kinscherf R (2012) Similar changes of gene expression in human skeletal muscle after resistance exercise and multiple fine needle biopsies. J Appl Physiol 112:289–295PubMedCrossRefGoogle Scholar
  26. Glass DJ (2003) Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol 5:87–90PubMedCrossRefGoogle Scholar
  27. Goldberg AL (1967) Work-induced growth of skeletal muscle in normal and hypophysectomized rats. Am J Physiol 213:1193–1198PubMedGoogle Scholar
  28. Goldberg AL, Goodman HM (1969) Amino acid transport during work-induced growth of skeletal muscle. Am J Physiol 216:1111–1115PubMedGoogle Scholar
  29. Goldspink DF (1977) The influence of immobilization and stretch on protein turnover of rat skeletal muscle. J Physiol 264:267–282PubMedCentralPubMedGoogle Scholar
  30. Goldspink G (2005) Mechanical signals, IGF-I gene splicing, and muscle adaptation. Physiology (Bethesda) 20:232–238CrossRefGoogle Scholar
  31. Goldspink DF, Cox VM, Smith SK, Eaves LA, Osbaldeston NJ, Lee DM, Mantle D (1995) Muscle growth in response to mechanical stimuli. Am J Physiol 268:E288–E297PubMedGoogle Scholar
  32. Gumucio JP, Mendias CL (2012) Atrogin-1, MuRF-1, and sarcopenia. Endocrine 43(1):12–21PubMedCentralPubMedCrossRefGoogle Scholar
  33. Henriksen EJ, Schneider MC, Ritter LS (1993) Regulation of contraction-stimulated system A amino acid uptake in skeletal muscle: role of vicinal sulfhydryls. Metabolism 42:440–445PubMedCrossRefGoogle Scholar
  34. Hubatsch DA, Jasmin BJ (1997) Mechanical stimulation increases expression of acetylcholinesterase in cultured myotubes. Am J Physiol 273:C2002–C2009PubMedGoogle Scholar
  35. Iwata M, Hayakawa K, Murakami T, Naruse K, Kawakami K, Inoue-Miyazu M, Yuge L, Suzuki S (2007) Uniaxial cyclic stretch-stimulated glucose transport is mediated by a ca-dependent mechanism in cultured skeletal muscle cells. Pathobiology 74:159–168PubMedCrossRefGoogle Scholar
  36. James PL, Stewart CE, Rotwein P (1996) Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism. J Cell Biol 133:683–693PubMedCrossRefGoogle Scholar
  37. Kandarian SC, Schulte LM, Esser KA (1992) Age effects on myosin subunit and biochemical alterations with skeletal muscle hypertrophy. J Appl Physiol 72:1934–1939PubMedGoogle Scholar
  38. Kim JS, Kosek DJ, Petrella JK, Cross JM, Bamman MM (2005) Resting and load-induced levels of myogenic gene transcripts differ between older adults with demonstrable sarcopenia and young men and women. J Appl Physiol 99:2149–2158PubMedCrossRefGoogle Scholar
  39. Lenk K, Schuler G, Adams V (2010) Skeletal muscle wasting in cachexia and sarcopenia: molecular pathophysiology and impact of exercise training. J Cachex Sarcopenia Muscle 1:9–21CrossRefGoogle Scholar
  40. Lewis MP, Tippett HL, Sinanan AC, Morgan MJ, Hunt NP (2000) Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures. J Muscle Res Cell Motil 21:223–233PubMedCrossRefGoogle Scholar
  41. Linderman JK, Talmadge RJ, Gosselink KL, Tri PN, Roy RR, Grindeland RE (1996) Synergistic ablation does not affect atrophy or altered myosin heavy chain expression in the non-weight bearing soleus muscle. Life Sci 59:789–795PubMedCrossRefGoogle Scholar
  42. Liu X, Lee DJ, Skittone LK, Natsuhara K, Kim HT (2010) Role of gelatinases in disuse-induced skeletal muscle atrophy. Muscle Nerve 41:174–178PubMedGoogle Scholar
  43. Louis E, Raue U, Yang Y, Jemiolo B, Trappe S (2007) Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle. J Appl Physiol 103:1744–1751PubMedCrossRefGoogle Scholar
  44. Martin NR, Passey SL, Player DJ, Khodabukus A, Ferguson RA, Sharples AP, Mudera V, Baar K, Lewis MP (2013) Factors affecting the structure and maturation of human tissue engineered skeletal muscle. Biomaterials 34:5759–5765PubMedCrossRefGoogle Scholar
  45. Matheny RW, Merritt E, Zannikos SV, Farrar RP, Adamo ML (2009) Serum IGF-I-deficiency does not prevent compensatory skeletal muscle hypertrophy in resistance exercise. Exp Biol Med 234:164–170CrossRefGoogle Scholar
  46. McCarthy JJ, Esser KA (2007) Counterpoint: satellite cell addition is not obligatory for skeletal muscle hypertrophy. J Appl Physiol 103:1100–1103PubMedCrossRefGoogle Scholar
  47. McKoy G, Ashley W, Mander J, Yang SY, Williams N, Russell B, Goldspink G (1999) Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation. J Physiol 516(Pt 2):583–592PubMedCentralPubMedCrossRefGoogle Scholar
  48. McPherron AC, Lee SJ (1997) Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 94:12457–12461PubMedCentralPubMedCrossRefGoogle Scholar
  49. McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387:83–90PubMedCrossRefGoogle Scholar
  50. Mehan RS, Greybeck BJ, Emmons K, Byrnes WC, Allen DL (2011) Matrix metalloproteinase-9 deficiency results in decreased fiber cross-sectional area and alters fiber type distribution in mouse hindlimb skeletal muscle. Cells Tissues Organs 194:510–520PubMedCentralPubMedCrossRefGoogle Scholar
  51. Mudera VC, Pleass R, Eastwood M, Tarnuzzer R, Schultz G, Khaw P, McGrouther DA, Brown RA (2000) Molecular responses of human dermal fibroblasts to dual cues: contact guidance and mechanical load. Cell Motil Cytoskelet 45:1–9CrossRefGoogle Scholar
  52. Mudera V, Smith AS, Brady MA, Lewis MP (2010) The effect of cell density on the maturation and contractile ability of muscle derived cells in a 3D tissue-engineered skeletal muscle model and determination of the cellular and mechanical stimuli required for the synthesis of a postural phenotype. J Cell Physiol 225:646–653PubMedCrossRefGoogle Scholar
  53. Nagase H, Woessner JF Jr (1999) Matrix metalloproteinases. J Biol Chem 274:21491–21494PubMedCrossRefGoogle Scholar
  54. Narici MV, Maffulli N (2010) Sarcopenia: characteristics, mechanisms and functional significance. Br Med Bull 95:139–159PubMedCrossRefGoogle Scholar
  55. O’Connor RS, Pavlath GK (2007) Point:Counterpoint: satellite cell addition is/is not obligatory for skeletal muscle hypertrophy. J Appl Physiol 103:1099–1100PubMedCrossRefGoogle Scholar
  56. Passey S, Martin N, Player D, Lewis MP (2011) Stretching skeletal muscle in vitro: does it replicate in vivo physiology? Biotechnol Lett 33(8):1513–1521PubMedCrossRefGoogle Scholar
  57. Pillard F, Laoudj-Chenivesse D, Carnac G, Mercier J, Rami J, Riviere D, Rolland Y (2011) Physical activity and sarcopenia. Clin Geriatr Med 27:449–470PubMedCrossRefGoogle Scholar
  58. Powell CA, Smiley BL, Mills J, Vandenburgh HH (2002) Mechanical stimulation improves tissue-engineered human skeletal muscle. Am J Physiol Cell Physiol 283:C1557–C1565PubMedCrossRefGoogle Scholar
  59. Rantanen T, Volpato S, Ferrucci L, Heikkinen E, Fried LP, Guralnik JM (2003) Handgrip strength and cause-specific and total mortality in older disabled women: exploring the mechanism. J Am Geriatr Soc 51:636–641PubMedCrossRefGoogle Scholar
  60. Rehfeldt C, Renne U, Sawitzky M, Binder G, Hoeflich A (2010) Increased fat mass, decreased myofiber size, and a shift to glycolytic muscle metabolism in adolescent male transgenic mice overexpressing IGFBP-2. Am J Physiol Endocrinol Metab 299:E287–E298PubMedGoogle Scholar
  61. Riikonen T, Westermarck J, Koivisto L, Broberg A, Kahari VM, Heino J (1995) Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression. J Biol Chem 270:13548–13552PubMedCrossRefGoogle Scholar
  62. Rosenblatt JD, Parry DJ (1992) Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. J Appl Physiol 73:2538–2543PubMedGoogle Scholar
  63. Roth SM, Martel GF, Ferrell RE, Metter EJ, Hurley BF, Rogers MA (2003) Myostatin gene expression is reduced in humans with heavy-resistance strength training: a brief communication. Exp Biol Med (Maywood) 228:706–709Google Scholar
  64. Sandri M (2008) Signaling in muscle atrophy and hypertrophy. Physiology 23:160–170PubMedCrossRefGoogle Scholar
  65. Schiaffino S, Bormioli SP, Aloisi M (1972) Cell proliferation in rat skeletal muscle during early stages of compensatory hypertrophy. Virchows Arch B Cell Pathol 11:268–273PubMedGoogle Scholar
  66. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108PubMedCrossRefGoogle Scholar
  67. Severgnini S, Lowenthal DT, Millard WJ, Simmen FA, Pollock BH, Borst SE (1999) Altered IGF-I and IGFBPs in senescent male and female rats. J Gerontol A Biol Sci Med Sci 54:B111–B115PubMedCrossRefGoogle Scholar
  68. Sharples AP, Stewart CE (2011) Myoblast models of skeletal muscle hypertrophy and atrophy. Curr Opin Clin Nutr Metab Care 14:230–236PubMedCrossRefGoogle Scholar
  69. Sharples AP, Al-Shanti N, Stewart CE (2010) C2 and C2C12 murine skeletal myoblast models of atrophic and hypertrophic potential: relevance to disease and ageing? J Cell Physiol 225:240–250PubMedCrossRefGoogle Scholar
  70. Sharples AP, Player DJ, Martin NR, Mudera V, Stewart CE, Lewis MP (2012) Modelling in vivo skeletal muscle ageing in vitro using three-dimensional bioengineered constructs. Aging Cell 11:986–995PubMedCrossRefGoogle Scholar
  71. Sharples AP, Al-Shanti N, Hughes DC, Lewis MP, Stewart CE (2013) The role of insulin-like-growth factor binding protein 2 (IGFBP2) and phosphatase and tensin homologue (PTEN) in the regulation of myoblast differentiation and hypertrophy. Growth Horm IGF Res 23:53–61PubMedCrossRefGoogle Scholar
  72. Smith AS, Passey S, Greensmith L, Mudera V, Lewis MP (2012) Characterization and optimization of a simple, repeatable system for the long term in vitro culture of aligned myotubes in 3D. J Cell Biochem 113:1044–1053PubMedCrossRefGoogle Scholar
  73. Trendelenburg AU, Meyer A, Rohner D, Boyle J, Hatakeyama S, Glass DJ (2009) Myostatin reduces Akt/TORC1/p70S6 K signaling, inhibiting myoblast differentiation and myotube size. Am J Physiol Cell Physiol 296:C1258–C1270PubMedCrossRefGoogle Scholar
  74. van den Beld AW, Blum WF, Pols HA, Grobbee DE, Lamberts SW (2003) Serum insulin-like growth factor binding protein-2 levels as an indicator of functional ability in elderly men. Eur J Endocrinol 148:627–634PubMedCrossRefGoogle Scholar
  75. Vandenburgh HH (1988) A computerized mechanical cell stimulator for tissue culture: effects on skeletal muscle organogenesis. In Vitro Cell Dev Biol 24:609–619PubMedCrossRefGoogle Scholar
  76. Vandenburgh H, Kaufman S (1979) In vitro model for stretch-induced hypertrophy of skeletal muscle. Science 203:265–268PubMedCrossRefGoogle Scholar
  77. Wang Q, McPherron AC (2012) Myostatin inhibition induces muscle fibre hypertrophy prior to satellite cell activation. J Physiol 590:2151–2165PubMedCentralPubMedGoogle Scholar
  78. Welle SL (2009) Myostatin and muscle fiber size. Focus on “Smad2 and 3 transcription factors control muscle mass in adulthood” and “Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size”. Am J Physiol Cell Physiol 296:C1245–C1247PubMedCrossRefGoogle Scholar
  79. Wolf E, Schneider MR, Zhou R, Fisch TM, Herbach N, Dahlhoff M, Wanke R, Hoeflich A (2005) Functional consequences of IGFBP excess-lessons from transgenic mice. Pediatr Nephrol 20:269–278PubMedCrossRefGoogle Scholar
  80. Yang Y, Jemiolo B, Trappe S (2006) Proteolytic mRNA expression in response to acute resistance exercise in human single skeletal muscle fibers. J Appl Physiol 101:1442–1450PubMedCrossRefGoogle Scholar
  81. Zanchi NE, de Siqueira Filho MA, Lira FS, Rosa JC, Yamashita AS, de Oliveira Carvalho CR, Seelaender M, Lancha-Jr AH (2009) Chronic resistance training decreases MuRF-1 and Atrogin-1 gene expression but does not modify Akt, GSK-3beta and p70S6K levels in rats. Eur J Appl Physiol 106:415–423PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • D. J. Player
    • 1
  • N. R. W. Martin
    • 1
  • S. L. Passey
    • 1
    • 4
  • A. P. Sharples
    • 2
    • 1
  • V. Mudera
    • 3
  • M. P. Lewis
    • 1
    Email author
  1. 1.Musculoskeletal Biology Research Group, School of Sport, Exercise and Health SciencesLoughborough UniversityLeicestershireUK
  2. 2.Stem Cells, Ageing and Molecular Physiology (SCAMP) Unit, Research Institute for Sport and Exercise Sciences (RISES), School of Sport and Exercise SciencesLiverpool John Moores UniversityLiverpoolUK
  3. 3.Division of Surgery and Interventional SciencesUCL Institute of Orthopaedics and Musculoskeletal ScienceStanmoreUK
  4. 4.Department of PharmacologyUniversity of MelbourneMelbourneAustralia

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