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Determining time of death: temperature-dependent postmortem changes in calcineurin A, MARCKS, CaMKII, and protein phosphatase 2A in mouse

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Abstract

While the determination of postmortem interval (PMI) is a crucial and fundamental step in any death investigation, the development of appropriate biochemical methods for PMI estimation is still in its infancy. This study focused on the temperature-dependent postmortem degradation of calcineurin A (CnA), calmodulin-dependent kinase II (CaMKII), myristoylated alanine-rich C-kinase substrate (MARCKs), and protein phosphatase 2A (PP2A) in mice. The results show that MARCKS, CaMKII, and the use of lung tissue do not appear to warrant further study for the determination of PMI in humans. In skeletal muscle, CnA underwent a rapid temperature-dependent cleavage (60 → 57 kDa) over the first 48 h of postmortem interval. At 21°C, this transformation was completed within 24 h. In contrast, PP2A increased within the first 24 h after which it degraded at 21°C but remained stable for up to 96 h at 5°C and 10°C. The 60 → 57 kDa postmortem conversion of CnA was inhibited by addition of protease inhibitors and MDL-28170 indicating a calpain pathway mediates this breakdown. Proteasome inhibition (MG-132) and calmodulin antagonism (calmidazolium) also inhibited this conversion suggesting that other protein degradation pathways also are in play. In contrast, all of the protease inhibitors and calmidazolium but not ethylene glycol tetraacetic acid led to increased levels of PP2A. The data are discussed in terms of developing a useable field-based biochemical assay for postmortem interval determination in humans and understanding the protein degradation pathways that are initiated upon death.

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Acknowledgments

We thank Andrew Catalano for his assistance with the dissection, Xavier D’Souza for assistance with animal care and maintenance, and Michael D. Rennie for assistance with the statistics. This project was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

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Authors and Affiliations

  1. Department of Cell & Systems Biology, University of Toronto, Toronto, ON, M5S 3G5, Canada

    Yekaterina O. Poloz & Danton H. O’Day

  2. Department of Biology, Forensic Science Program, University of Toronto at Mississauga, 3359 Mississauga Rd., Mississauga, ON, L5L 1C6, Canada

    Danton H. O’Day

Authors
  1. Yekaterina O. Poloz
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  2. Danton H. O’Day
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Correspondence to Danton H. O’Day.

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Poloz, Y.O., O’Day, D.H. 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 (2009). https://doi.org/10.1007/s00414-009-0343-x

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  • DOI: https://doi.org/10.1007/s00414-009-0343-x

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