Skip to main content

Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death

Abstract

Estimating the time since death is a very important aspect in forensic sciences which is pursued by a variety of methods. The most precise method to determine the postmortem interval (PMI) is the temperature method which is based on the decrease of the body core temperature from 37 °C. However, this method is only useful in the early postmortem phase (~0–36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyze the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 h postmortem (hpm). Our results demonstrate that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated. Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently. This degradation behavior identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. 1.

    Henssge C, Madea B (2007) Estimation of the time since death. Forensic Sci Int 165:182–184

    Article  PubMed  Google Scholar 

  2. 2.

    Madea B (1994) Importance of supravitality in forensic medicine. Forensic Sci Int 69:221–241

    Article  CAS  PubMed  Google Scholar 

  3. 3.

    Henssge C, Althaus L, Bolt J, Freislederer A, Haffner HT, Henssge CA, Hoppe B, Schneider V (2000) Experiences with a compound method for estimating the time since death. I. Rectal temperature nomogram for time since death. Int J Legal Med 113:303–319

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Henssge C, Althaus L, Bolt J, Freislederer A, Haffner HT, Henssge CA, Hoppe B, Schneider V (2000) Experiences with a compound method for estimating the time since death. II. Integration of non-temperature-based methods. Int J Legal Med 113:320–331

    Article  CAS  PubMed  Google Scholar 

  5. 5.

    Muñoz Barús JI, Suárez-Peñaranda J, Otero XL, Rodríguez-Calvo MS, Costas E, Miguéns X, Concheiro L (2002) Improved estimation of postmortem interval based on differential behaviour of vitreous potassium and hypoxantine in death by hanging. Forensic Sci Int 125:67–74

    Article  PubMed  Google Scholar 

  6. 6.

    Jackson ARW, Jackson JM (2011) Forensic science.In: Pearson Education Limited, 3rd edn. pp 376–381

  7. 7.

    Kimura A, Ishida Y, Hayashi T, Nosaka M, Kondo T (2010) Estimating time of death based on the biological clock. Int J Legal Med 125:385–391

    Article  PubMed  Google Scholar 

  8. 8.

    Amendt J, Richards CS, Campobasso CP, Zehner R, Hall MJR (2011) Forensic entomology: applications and limitations. Forensic Sci Med Pathol 7:379–392

    Article  CAS  PubMed  Google Scholar 

  9. 9.

    Metcalf JL, Wegener Parfrey L, Gonzalez A, Lauber CL, Knights D, Ackermann G, Humphrey GC, Gebert MJ, Van Treuren W, Berg-Lyons D, Keepers K, Guo Y, Bullard J, Fierer N, Carter DO, Knight R (2013) A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system. elife 2(e01104):1–19

    Google Scholar 

  10. 10.

    Young ST, Wells JD, Hobbs GR, Bishop CP et al (2013) Estimating postmortem interval using RNA degradation and morphological changes in tooth pulp. Forensic Sci Int 229:163.e1–163.e6

    Article  CAS  Google Scholar 

  11. 11.

    Itani M, Yamamoto Y, Doi Y, Miyaishi S (2011) Quantitative analysis of DNA degradation in the dead body. Acta Med Okayama 65:299–306

    CAS  PubMed  Google Scholar 

  12. 12.

    Alibegović A (2014) Cartilage: a new parameter for the determination of the postmortem interval? J Forensic Legal Med 27:39–45

    Article  Google Scholar 

  13. 13.

    Tomita Y, Nihira M, Ohno Y, Sato S (2004) Ultrastructural changes during in situ early postmortem autolysis in kidney, pancreas, liver, heart and skeletal muscle of rats. Legal Med 6:25–31

    Article  PubMed  Google Scholar 

  14. 14.

    Collan Y, Salmenperä M (1976) Electron microscopy of postmortem autolysis of rat muscle tissue. Acta Neuropathol 35:219–233

    CAS  PubMed  Google Scholar 

  15. 15.

    Tokunaga I, Takeichi S, Yamamoto A, Gotoda M, Maeiwa M (1993) Comparison of postmortem autolysis in cardiac and skeletal muscle. J Forensic Sci 38:1187–1193

    Article  CAS  PubMed  Google Scholar 

  16. 16.

    Kang S, Kassam N, Gauthier ML, O'Day DH (2003) Post-mortem changes in calmodulin binding proteins in muscle and lung. Forensic Sci Int 131:140–147

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Poloz YO, O'Day DH (2009) Determining time of death: temperature-dependent postmortem changes in calcineurin A, MARCKS, CaMKII, and protein phosphatase 2A in mouse. Int J Legal Med 123:305–314

    Article  PubMed  Google Scholar 

  18. 18.

    Sanoudou D, Kang PB, Haslett JN, Han M, Kunkel LM, Beggs AH (2004) Transcriptional profile of postmortem skeletal muscle. Physiol Genomics 16:222–228

    Article  CAS  PubMed  Google Scholar 

  19. 19.

    Taylor RG, Geesink GH, Thompson VF, Koohmaraie M, Goll DE (1995) Is Z-disk degradation responsible for postmortem tenderization? J Anim Sci 73:1351–1367

    CAS  PubMed  Google Scholar 

  20. 20.

    Huff-Lonergan E, Mitsuhashi T, Beekman DD, Parrish FC Jr, Olson DG, Robson RM (1996) Proteolysis of specific muscle structural proteins by mu-calpain at low pH and temperature is similar to degradation in postmortem bovine muscle. J Anim Sci 74:993–1008

    CAS  PubMed  Google Scholar 

  21. 21.

    Huff-Lonergan E, Mitsuhashi T, Parrish FC Jr, Robson RM (1996) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting comparisons of purified myofibrils and whole muscle preparations for evaluating titin and nebulin in postmortem bovine muscle. J Anim Sci 74:779–785

    CAS  PubMed  Google Scholar 

  22. 22.

    Tomaszewska-Gras J, Kijowski J, Schreurs FJ (2002) Quantitative determination of titin and nebulin in poultry meat by SDS-PAGE with an internal standard. Meat Sci 62:61–66

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Szalata M, Pospiech E, Greaser ML, Lyczynski A, Grzes B, Mikolajczak B (2005) Titin and troponin T changes in relation to tenderness of meat from pigs of various meatiness. Pol J Food Nutr Sci 14:139–144

    CAS  Google Scholar 

  24. 24.

    Wu G, Clerens S, Farouk MM (2014) LC MS/MS identification of large structural proteins from bull muscle and their degradation products during post mortem storage. Food Chem 150:137–144

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    Wu G, Farouk MM, Clerens S, Rosenvold K (2014) Effect of beef ultimate pH and large structural protein changes with aging on meat tenderness. Meat Sci 98:637–645

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    Tomaszewska-Gras J, Schreurs FJ, Kijowski J (2011) Post mortem development of meat quality as related to changes in cytoskeletal proteins of chicken muscles. Br Poult Sci 52:189–201

    Article  CAS  PubMed  Google Scholar 

  27. 27.

    Melody JL, Lonergan SM, Rowe LJ, Huiatt TW, Mayes MS, Huff-Lonergan E (2004) Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles. J Anim Sci 82:1195–1205

    CAS  PubMed  Google Scholar 

  28. 28.

    Matsuura T, Kimura S, Ohtsuka S, Maruyama K (1991) Isolation and characterization of 1,200 kDa peptide of alpha-connectin. J Biochem 110:474–478

    CAS  PubMed  Google Scholar 

  29. 29.

    Warren CM, Krzesinski PR, Greaser ML (2003) Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins. Electrophoresis 24:1695–1702

    Article  CAS  PubMed  Google Scholar 

  30. 30.

    Koohmaraie M, Geesink GH (2006) Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system. Meat Sci 74:34–43

    Article  CAS  PubMed  Google Scholar 

  31. 31.

    Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system. Physiol Rev 83:731–801

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Pomponio L, Ertbjerg P (2012) The effect of temperature on the activity of μ- and m-calpain and calpastatin during post-mortem storage of porcine longissimus muscle. Meat Sci 91:50–55

    Article  CAS  PubMed  Google Scholar 

  33. 33.

    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  34. 34.

    Raser KJ, Posner A, Wang KK (1995) Casein zymography: a method to study mu-calpain, m-calpain, and their inhibitory agents. Arch Biochem Biophys 319:211–216

    Article  CAS  PubMed  Google Scholar 

  35. 35.

    Meyer LC, Wright NT (2013) Structure of giant muscle proteins. Physiol 4 Article 368:1–12

    Google Scholar 

  36. 36.

    Huff Lonergan E, Zhang W, Lonergan SM (2010) Biochemistry of postmortem muscle - lessons on mechanisms of meat tenderization. Meat Sci 86:184–195

    Article  CAS  PubMed  Google Scholar 

  37. 37.

    Ho CY, Stromer MH, Rouse G, Robson RM (1997) Effects of electrical stimulation and postmortem storage on changes in titin, nebulin, desmin, troponin-T, and muscle ultrastructure in Bos indicus crossbred cattle. J Anim Sci 75:366–376

    CAS  PubMed  Google Scholar 

  38. 38.

    Geesink GH, Bekhit AD, Bickerstaffe R (2000) Rigor temperature and meat quality characteristics of lamb longissimus muscle. J Anim Sci 78:2842–2848

    CAS  PubMed  Google Scholar 

  39. 39.

    Koohmaraie M, Shackelford SD, Wheeler TL, Lonergan SM, Doumit ME (1995) A muscle hypertrophy condition in lamb (callipyge): characterization of effects on muscle growth and meat quality traits. J Anim Sci 73:3596–3607

    CAS  PubMed  Google Scholar 

  40. 40.

    Rowe LJ, Maddock KR, Lonergan SM, Huff-Lonergan E (2004) Oxidative environments decrease tenderization of beef steaks through inactivation of μ-calpain. J Anim Sci 82:3254–3266

    CAS  PubMed  Google Scholar 

  41. 41.

    Zhang WG, Lonergan SM, Gardner MA, Huff-Lonergan E (2006) Contribution of postmortem changes of integrin, desmin and μ-calpain to variation in water holding capacity of pork. Meat Sci 74:578–585

    Article  CAS  PubMed  Google Scholar 

  42. 42.

    Geesink GH, Koohmaraie M (1999) Postmortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb biceps femoris during extended postmortem storage. J Anim Sci 77:1490–1501

    CAS  PubMed  Google Scholar 

  43. 43.

    Baron CP, Jacobsen S, Purslow PP (2004) Cleavage of desmin by cysteine proteases: Calpains and cathepsin B. Meat Sci 68:447–456

    Article  CAS  PubMed  Google Scholar 

  44. 44.

    Bodor GS, Survant L, Voss EM, Smith S, Porterfield D, Apple FS (1997) Cardiac troponin T composition in normal and regenerating human skeletal muscle. Clin Chem 43:476–484

    CAS  PubMed  Google Scholar 

  45. 45.

    Veiseth E, Shackelford SD, Wheeler TL, Koohmaraie M (2001) Effect of postmortem storage on μ-calpain and m-calpain in ovine skeletal muscle. J Anim Sci 79:1502–1508

    CAS  PubMed  Google Scholar 

  46. 46.

    Boehm ML, Kendall TL, Thompson VF, Goll DE (1998) Changes in the calpains and calpastatin during postmortem storage of bovine muscle. J Anim Sci 76:2415–2434

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors are grateful to Fiona Bergmann, Magdalena Brandauer, and Christian Platzl for providing excellent technical assistance, as well as Roman Fuchs and Arne Bathke for advices and assistance in statistics. Our gratitude also goes to Elena Esra Foditsch and Edith Tutsch-Bauer for exchange of ideas and discussion throughout the study.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Peter Steinbacher.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pittner, S., Monticelli, F.C., Pfisterer, A. et al. Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death. Int J Legal Med 130, 421–431 (2016). https://doi.org/10.1007/s00414-015-1210-6

Download citation

Keywords

  • Postmortem interval (PMI)
  • Protein
  • Degradation
  • Skeletal muscle
  • Pig