European Journal of Applied Physiology

, Volume 103, Issue 3, pp 323–332 | Cite as

Effects of concentric and repeated eccentric exercise on muscle damage and calpain–calpastatin gene expression in human skeletal muscle

  • Kristian Vissing
  • Kristian Overgaard
  • Anders Nedergaard
  • Anne Fredsted
  • Peter Schjerling
Original Article


The purpose of this study was to compare the responsiveness of changes in Ca2+-content and calpain–calpastatin gene expression to concentric and eccentric single-bout and repeated exercise. An exercise group (n = 14) performed two bouts of bench-stepping exercise with 8 weeks between exercise bouts, and was compared to a control-group (n = 6). Muscle strength and soreness and plasma creatine kinase and myoglobin were measured before and during 7 days following exercise bouts. Muscle biopsies were collected from m. vastus lateralis of both legs prior to and at 3, 24 h and 7 days after exercise and quantified for muscle Ca2+-content and mRNA levels for calpain isoforms and calpastatin. Exercise reduced muscle strength and increased muscle soreness predominantly in the eccentric leg (P < 0.05). These responses as well as plasma levels of creatine kinase and myoglobin were all attenuated after the repeated eccentric exercise bout (P < 0.05). Total muscle Ca2+-content did not differ between interventions. mRNA levels for calpain 2 and calpastatin were upregulated exclusively by eccentric exercise 24 h post-exercise (P < 0.05), with no alteration in expression between bouts. Calpain 1 and calpain 3 mRNA did not change at any specific time point post-exercise for either intervention. Our mRNA results suggest a regulation on the calpain–calpastatin expression response to muscle damaging eccentric exercise, but not concentric exercise. Although a repeated bout effect was demonstrated in terms of muscle function, no immediate support was provided to suggest that regulation of expression of specific system components is involved in the repeated bout adaptation.


Calcium proteolysis Repeated bout effect Single-bout Transcriptional regulation 



We wish to acknowledge the Danish Health Research Agency (grants no. 22-04-0454), the Ministry of Culture (grant no. 2004-05-029), the NovoNordisk Foundation, Hovedstadens Sygehusfaellesskab and the Medical Faculty at the University of Copenhagen. Furthermore, we would like to thank Thorsten Ingemann Hansen for clinical assistance and Anne Mette Kloster for technical assistance.

Supplementary material

421_2008_709_MOESM1_ESM.ppt (525 kb)
ESM1 (PPT 525 kb)


  1. Arendt-Nielsen L (2002) Clinical use of pain measurement techniques. Ugeskr Laeger 164:1790–1795PubMedGoogle Scholar
  2. Belcastro AN (1993) Skeletal muscle calcium-activated neutral protease (calpain) with exercise. J Appl Physiol 74:1381–1386PubMedGoogle Scholar
  3. Belcastro AN, Shewchuk LD, Raj DA (1998) Exercise-induced muscle injury: a calpain hypothesis. Mol Cell Biochem 179:135–145PubMedCrossRefGoogle Scholar
  4. Busquets S, Garcia-Martinez C, Alvarez B, Carbo N, Lopez-Soriano FJ, Argiles JM (2000) Calpain-3 gene expression is decreased during experimental cancer cachexia. Biochim Biophys Acta 1475:5–9PubMedGoogle Scholar
  5. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. doi: 10.1006/abio.1987.9999 PubMedCrossRefGoogle Scholar
  6. Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GA, Zumla A (2005) The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344:141–143. doi: 10.1016/j.ab.2005.05.022 PubMedCrossRefGoogle Scholar
  7. Dietrichson P, Coakley J, Smith PE, Griffiths RD, Helliwell TR, Edwards RH (1987) Conchotome and needle percutaneous biopsy of skeletal muscle. J Neurol Neurosurg Psychiatry 50:1461–1467PubMedGoogle Scholar
  8. Edwards RH, Young A, Hosking GP, Jones DA (1977) Human skeletal muscle function: description of tests and normal values. Clin Sci Mol Med 52:283–290PubMedGoogle Scholar
  9. Feasson L, Stockholm D, Freyssenet D, Richard I, Duguez S, Beckmann JS, Denis C (2002) Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric exercise in human skeletal muscle. J Physiol 543:297–306PubMedCrossRefGoogle Scholar
  10. Fluck M, Dapp C, Schmutz S, Wit E, Hoppeler H (2005) Transcriptional profiling of tissue plasticity: role of shifts in gene expression and technical limitations. J Appl Physiol 99:397–413. doi: 10.1152/japplphysiol.00050.2005 PubMedCrossRefGoogle Scholar
  11. Friden J, Sjostrom M, Ekblom B (1983) Myofibrillar damage following intense eccentric exercise in man. Int J Sports Med 4:170–176PubMedCrossRefGoogle Scholar
  12. Gissel H, Clausen T (1999) Excitation-induced Ca2+ uptake in rat skeletal muscle. Am J Physiol 276:R331–R339PubMedGoogle Scholar
  13. Gissel H, Clausen T (2001) Excitation-induced Ca2+ influx and skeletal muscle cell damage. Acta Physiol Scand 171:327–334PubMedCrossRefGoogle Scholar
  14. Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system. Physiol Rev 83:731–801. doi: 10.1152/physrev.00029.2002 PubMedGoogle Scholar
  15. Huang J, Forsberg NE (1998) Role of calpain in skeletal-muscle protein degradation. Proc Natl Acad Sci USA 95:12100–12105PubMedCrossRefGoogle Scholar
  16. Jackman RW, Kandarian SC (2004) The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol 287:C834–C843. doi: 10.1152/ajpcell.00579.2003 PubMedCrossRefGoogle Scholar
  17. Jonsdottir IH, Schjerling P, Ostrowski K, Asp S, Richter EA, Pedersen BK (2000) Muscle contractions induce interleukin-6 mRNA production in rat skeletal muscles. J Physiol 528(Pt 1):157–163PubMedCrossRefGoogle Scholar
  18. Kinbara K, Ishiura S, Tomioka S, Sorimachi H, Jeong SY, Amano S, Kawasaki H, Kolmerer B, Kimura S, Labeit S, Suzuki K (1998) Purification of native p94, a muscle-specific calpain, and characterization of its autolysis. Biochem J 335(Pt 3):589–596PubMedGoogle Scholar
  19. Konig N, Raynaud F, Feane H, Durand M, Mestre-Frances N, Rossel M, Ouali A, Benyamin Y (2003) Calpain 3 is expressed in astrocytes of rat and Microcebus brain. J Chem Neuroanat 25:129–136PubMedCrossRefGoogle Scholar
  20. Larsen RG, Ringgaard S, Overgaard K (2007) Localization and quantification of muscle damage by magnetic resonance imaging following step exercise in young women. Scand J Med Sci Sports 17:76–83. doi: 10.1111/j.1600-0838.2006.00525.x PubMedGoogle Scholar
  21. Mahoney DJ, Carey K, Fu MH, Snow R, Cameron-Smith D, Parise G, Tarnopolsky MA (2004) Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise. Physiol Genomics 18:226–231. doi: 10.1152/physiolgenomics.00067.2004 PubMedCrossRefGoogle Scholar
  22. Malm C (2001) Exercise-induced muscle damage and inflammation: fact or fiction? Acta Physiol Scand 171:233–239PubMedCrossRefGoogle Scholar
  23. McHugh MP (2003) Recent advances in the understanding of the repeated bout effect: the protective effect against muscle damage from a single bout of eccentric exercise. Scand J Med Sci Sports 13:88–97PubMedCrossRefGoogle Scholar
  24. Murphy RM, Goodman CA, McKenna MJ, Bennie J, Leikis M, Lamb GD (2007) Calpain-3 is autolyzed and hence activated in human skeletal muscle 24 h following a single bout of eccentric exercise. J Appl Physiol 103:926–931. doi: 10.1152/japplphysiol.01422.2006 PubMedCrossRefGoogle Scholar
  25. Nedergaard A, Vissing K, Overgaard K, Kjaer M, Schjerling P (2007) Expression patterns of atrogenic and ubiquitin proteasome component genes with exercise: effect of different loading patterns and repeated exercise bouts. J Appl Physiol 103:1513–1522. doi: 10.1152/japplphysiol.01445.2006 PubMedCrossRefGoogle Scholar
  26. Newham DJ, Jones DA, Edwards RH (1983) Large delayed plasma creatine kinase changes after stepping exercise. Muscle Nerve 6:380–385. doi: 10.1002/mus.880060507 PubMedCrossRefGoogle Scholar
  27. Nosaka K, Sakamoto K, Newton M, Sacco P (2001) How long does the protective effect on eccentric exercise-induced muscle damage last? Med Sci Sports Exerc 33:1490–1495PubMedCrossRefGoogle Scholar
  28. Nosaka K, Newton MJ, Sacco P (2005) Attenuation of protective effect against eccentric exercise-induced muscle damage. Can J Appl Physiol 30:529–542PubMedGoogle Scholar
  29. Overgaard K, Fredsted A, Hyldal A, Ingemann-Hansen T, Gissel H, Clausen T (2004) Effects of running distance and training on Ca2+ content and damage in human muscle. Med Sci Sports Exerc 36:821–829PubMedCrossRefGoogle Scholar
  30. Raynaud P, Gillard M, Parr T, Bardsley R, Amarger V, Leveziel H (2005a) Correlation between bovine calpastatin mRNA transcripts and protein isoforms. Arch Biochem Biophys 440:46–53. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  31. Raynaud P, Jayat-Vignoles C, Laforet MP, Leveziel H, Amarger V (2005b) Four promoters direct expression of the calpastatin gene. Arch Biochem Biophys 437:69–77. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  32. Schjerling P (2001) The importance of internal controls in mRNA quantification. J Appl Physiol 90:401–402PubMedGoogle Scholar
  33. Sorichter S, Mair J, Koller A, Gebert W, Rama D, Calzolari C, Artner-Dworzak E, Puschendorf B (1997) Skeletal troponin I as a marker of exercise-induced muscle damage. J Appl Physiol 83:1076–1082PubMedGoogle Scholar
  34. Sorimachi H, Toyama-Sorimachi N, Saido TC, Kawasaki H, Sugita H, Miyasaka M, Arahata K, Ishiura S, Suzuki K (1993) Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J Biol Chem 268:10593–10605PubMedGoogle Scholar
  35. Spencer MJ, Lu B, Tidball JG (1997) Calpain II expression is increased by changes in mechanical loading of muscle in vivo. J Cell Biochem 64:55–66. doi: 10.1002/(SICI)1097-4644(199701)64:1 PubMedCrossRefGoogle Scholar
  36. Stupka N, Tarnopolsky MA, Yardley NJ, Phillips SM (2001) Cellular adaptation to repeated eccentric exercise-induced muscle damage. J Appl Physiol 91:1669–1678PubMedGoogle Scholar
  37. Tidball JG, Spencer MJ (2002) Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse. J Physiol 545:819–828PubMedCrossRefGoogle Scholar
  38. Vissing K, Andersen JL, Schjerling P (2005) Are exercise-induced genes induced by exercise? FASEB J 19:94–96. doi: 10.1096/fj.04-2084fje PubMedGoogle Scholar
  39. Warren GL, Lowe DA, Armstrong RB (1999) Measurement tools used in the study of eccentric contraction-induced injury. Sports Med 27:43–59PubMedCrossRefGoogle Scholar
  40. Wei W, Fareed MU, Evenson A, Menconi MJ, Yang H, Petkova V, Hasselgren PO (2005) Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3. Am J Physiol Regul Integr Comp Physiol 288:R580–R590. doi: 10.1152/ajpregu.00341.2004 PubMedGoogle Scholar
  41. Williams AB, Decourten-Myers GM, Fischer JE, Luo G, Sun X, Hasselgren PO (1999) Sepsis stimulates release of myofilaments in skeletal muscle by a calcium-dependent mechanism. FASEB J 13:1435–1443PubMedGoogle Scholar
  42. Yeung EW, Allen DG (2004) Stretch-activated channels in stretch-induced muscle damage: role in muscular dystrophy. Clin Exp Pharmacol Physiol 31:551–556. doi: 10.1111/j.1440-1681.2004.04027.x PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Kristian Vissing
    • 1
  • Kristian Overgaard
    • 1
  • Anders Nedergaard
    • 2
    • 4
  • Anne Fredsted
    • 3
  • Peter Schjerling
    • 4
    • 5
  1. 1.Department of Sport ScienceUniversity of AarhusAarhus CDenmark
  2. 2.Institute of Sports MedicineBispebjerg HospitalCopenhagenDenmark
  3. 3.Institute of Physiology and BiophysicsUniversity of AarhusAarhusDenmark
  4. 4.Department of Molecular Muscle BiologyCopenhagen Muscle Research CentreCopenhagenDenmark
  5. 5.Department of Medical Biochemistry and GeneticsUniversity of CopenhagenCopenhagenDenmark

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