Post-mortem changes in calmodulin binding proteins in muscle and lung

Abstract

Estimation of post-mortem interval (PMI) remains an elusive issue in forensic investigations. In this study, we examined the possible use of calmodulin (CaM) binding proteins (CaMBPs) as indicators of PMI. Whole CaMBP populations from homogenized rat lung and rat skeletal muscle removed at 0, 24, 48 and 96h post-mortem at 21°C were detected by the calmodulin binding overlay technique (CaMBOT) using 35S-VU1-CaM and visualized by autoradiography. CaMBOT showed that, in both tissues, the CaMBP population remained relatively stable for up to 96h post-mortem with the exception of a single ∼200kDa CaMBP that increased in 24h post-mortem samples then showed decreasing amounts at subsequent times. Immunoblot analysis of the specific CaMBPs, Ca2+/CaM-dependent kinase II (CaMKII), calcineurin A (CNA), myristoylated alanine-rich C-kinase substrate (MARCKS) and inducible nitric oxide synthase (iNOS) were done on lung tissue samples. CaMKII levels did not change appreciably over the 96h PMI examined. In contrast to iNOS levels, which varied from sample to sample, CNA and MARCKS showed predictable patterns of change: the level of MARCKS decreased steadily in the 0–96h post-mortem lung samples while CNA underwent a shift in mobility on SDS-PAGE by 24h post-mortem before slowly decreasing in amount. The stability of CaMKII levels over 96h was also seen in skeletal muscle tissue while CNA showed variable levels at 0, 48 and 96h with the presence of the rapidly migrating band at 24h. These patterns of change in CaMBPs provide some insight into the post-mortem changes in calmodulin-mediated signaling components in lung and skeletal muscle and support the further study of CNA and CaMKII as potential markers for estimating short- and long-term PMIs.