Protein degradation in human skeletal muscle: advancing postmortem interval estimation under natural outdoor conditions
Eileen Holzer, Jane C. Harris, Janine Brüderl, Katharina Helm, Laura Flachberger, Evan P. S. Pratt, Peter Steinbacher, Fabio Monticelli, Stefan Pittner
TL;DR
This study explores how proteins in human muscle degrade over time outdoors, aiming to improve postmortem interval estimation in forensic science.
Contribution
The study provides longitudinal human skeletal muscle proteomic data collected under natural outdoor conditions with environmental monitoring.
Findings
Protein degradation patterns in skeletal muscle are influenced by environmental conditions.
Specific proteins degrade in regular, predictable patterns under natural outdoor settings.
An extracorporeal model showed reproducible proteolytic trends comparable to in situ degradation.
Abstract
Estimating the postmortem interval (PMI) represents a major challenge in forensic science, particularly beyond the early postmortem phase. Protein degradation has become a promising molecular approach, as many proteins disintegrate in progressive and temporally distinct degradation patterns. However, decomposition is influenced by environmental and individual factors, resulting in case-specific variability that complicates the development of generally applicable PMI estimation methods. Available human reference data are limited and often derived from autopsy samples collected at a single time point postmortem, typically without precise PMI or environmental data. Longitudinal studies under monitored natural outdoor conditions are therefore essential to establish reliable reference data for protein-based PMI estimation. This study investigated postmortem protein degradation in skeletal…
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Figure 7- —Paris Lodron University of Salzburg
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Taxonomy
TopicsForensic Entomology and Diptera Studies · Forensic Toxicology and Drug Analysis · Forensic and Genetic Research
Introduction
Estimating the postmortem interval (PMI) is a central and challenging task in forensic science. After death, a cascade of physiological and biochemical changes occurs in the body, and phenomena such as algor mortis (body cooling), rigor mortis (muscle stiffening), and livor mortis (settling of blood) are commonly employed to estimate the time since death in early postmortem phases [1, 2]. However, the applicability of these methods is largely limited to the early PMI, and there is a critical lack of approaches for estimations in later postmortem stages [3]. Forensic entomology, which investigates the presence and developmental stage of necrophagous insects as indicators of the minimum PMI is a widely accepted and reliable method for the advanced postmortem phase [4]. Nevertheless, its application is restricted to the presence, or detection of entomological evidence, which is often hindered by temperature, weather, or accessibility [5–7]. Another approach is the morphological assessment of decomposition. Scoring systems such as the Total Body Score (TBS) by Megyesi et al. [8], that was later reviewed and improved by Moffatt et al. [9] quantify morphological changes of a body and are joined with accumulated degree days (ADD) to account for the temperature dependent progression of decomposition [5, 8, 9]. However, these scoring systems are subject to certain limitations, including variations in deposition environment and region-specific decomposition patterns [10]. Additionally, the decomposition process itself is influenced by numerous external and internal factors resulting in highly case-specific progression patterns that complicate the development of generally applicable models for PMI estimation [11, 12]. Therefore, it is required to validate the applicability of respective scoring methods and ADD calculation for different environments and regions.
In recent years, molecular approaches have been investigated as complementary methods for PMI estimation. The postmortem behavior of biomolecules such as DNA, RNA, microRNAs, lipids and proteins have been in focus to delimit the PMI based on (more or less) distinct degradation patterns [6, 13, 14]. Concordantly, other molecular approaches, such as microbiome-based analyses have demonstrated potential for PMI estimation in later stages. However, despite promising experimental findings, none of these approaches have yet to be routinely applied in forensic casework [6].
Among these approaches, protein degradation has proven to be especially valuable for PMI estimation. Different molecular properties of proteins account for their variable susceptibility to enzymatic degradation [15]. These characteristics allow for the selection of markers with defined temporal degradation patterns. Numerous studies have shown that specific proteins degrade in a predictable, time-dependent manner [6, 15–18].
Skeletal muscle is a particularly suitable tissue for such analyses because it is the most abundant tissue in the human body, relatively well protected by the skin and easily accessible [16]. A considerable challenge in applying the analysis of postmortem protein degradation in time since death estimation is the variability caused by intrinsic and extrinsic factors that influence the dynamics of the decomposition process. These are primarily driven by extrinsic conditions, although intrinsic factors such as age, body mass, medication and diseases can substantially alter the velocity of the degradation processes [6, 12]. Temperature is considered the most critical extrinsic factor, as it strongly affects the rate of proteolytic activity, accelerating degradation in warm environments and decelerating it under cold conditions. In addition, moisture promotes microbial proliferation and enzymatic processes, while insect activity can significantly accelerate tissue breakdown through mechanical disruption and insect-derived enzymes [6, 19, 20]. Given these multifactorial influences, there is a strong need for defined confidence intervals, exclusion criteria, correction factors and reference datasets to enable PMI estimation using protein-based approaches [6].
Previous research has mainly focused on animal models, particularly pigs, due to their anatomical and physiological similarities to humans [21]. Although animal models enable large sample sizes and controlled conditions, their applicability to humans remains limited [22–24]. In contrast, sample material derived from autopsy cases can only be obtained from a single time point postmortem and is associated with a multitude of (uncontrollable) potential influencing factors, making it virtually impossible to investigate the dynamics of degradation over time. Moreover, information regarding the precise PMI (and thus the target variable) is often lacking or incomplete [23]. Taken together, these limitations highlight the importance of longitudinal studies on human tissue under well documented environmental conditions [6].
Human taphonomy research facilities provide a unique opportunity to conduct such studies, but currently only a few locations exist worldwide. Most of them are located in the United States, with additional sites in Canada, Australia and the Netherlands [25]. One of these facilities is the Forensic Research Outdoor Station (FROST) in Marquette, Michigan affiliated with Northern Michigan University [25]. FROST is dedicated to research on human decomposition under natural outdoor conditions, allowing for longitudinal analyses of morphological and molecular changes in individuals with known PMI.
This study aims to investigate morphological decomposition as well as postmortem protein degradation in skeletal muscle under natural conditions at FROST. The progress of decomposition was monitored over a period of 8 days during two field trials (in 2022 and 2024) from a total of five whole body donors through regular assessment of morphological changes using the TBS [8, 9]. In the same intervals thigh muscle samples were collected to investigate postmortem protein degradation patterns. In addition to in situ sampling, an extracorporeal model was introduced, where an excised muscle block was stored in close proximity to the donors and samples collected simultaneously. This setup enabled a direct comparison of degradation patterns from tissue within and outside the body and served to evaluate the applicability of an extracorporeal setting as a standardized model for future investigations of potential influencing factors. Despite the small sample size and different environmental and individual influencing factors, this study provides first comparative data on degradation patterns under varying environmental conditions and supports the advancement of molecular markers for PMI estimation.
Materials and methods
Body donors and study design
A total of five human body donors was included in this study, with three placed at the outdoor facility in July 2022 and two in August 2024. Table 1 provides an overview of the donor characteristics. Prior to placement at the outdoor site, all donors were stored under constant freezing or under a combination of freezing and refrigeration (administrative procedures, the coordination (accumulation) of multiple donors, and seasonal scheduling led to pre-placement intervals of approximately 100–400 days).Table 1. Summary of donor demographic data and storage conditions. Age at death and PMI prior to placement are rounded to preserve anonymity of the donors. For the same reason medical information (including causes of death) cannot be disclosed here but can be inspected upon requestDonor ADonor BDonor CDonor DDonor EPlacementJuly 2022July 2022July 2022August 2024August 2024Age at death [years]7090857040SexMaleMaleFemaleMaleMaleHeight [cm]180193165183175Weight [kg]6164457199BMI18.817.216.521.232.3PMI prior to placement [days]100100200400150Storage conditionFreezerFreezerFreezer/refrigerationFreezer/refrigerationFreezer/refrigerationManner of deathNaturalNaturalNaturalNaturalNaturalCommentsPartially frozen at placementPartially frozen at placementPartially frozen at placement
All donors were exposed to natural outdoor conditions at the same field site and monitored over eight days (day 0 to day 7). In 2022, the bodies were positioned supine or prone on grass or on bare soil (due to a different experimental approach). In 2024, both individuals were positioned supine on grass. All bodies were naked and covered only with wire mesh cages to limit scavenging.
To investigate postmortem changes over time, both morphological decomposition and degradation of muscle proteins were documented and studied. For protein analysis, skeletal muscle tissue was collected from each donor once per day from two setups: in situ biopsies collected from the right thigh (M. vastus lateralis) and extracorporeal subsampling. For the latter, a muscle block of approximately 5 × 3 cm was excised from the left and transferred to a plastic container immediately following donor placement. This container was stored in a lightproof box together with a temperature data logger (testo 174 H).
All further observations and tissue collections were conducted in 24-h intervals starting from day 0, ensuring temporal comparability across donors and trials. Environmental parameters (i.e. air temperature, relative humidity and precipitation) were recorded in 15-min intervals by an on-site weather station.
Morphological assessment
Morphological changes of all body donors were assessed daily using the Total Body Score (TBS) by Megyesi et al. [8]. Despite flaws in their ADD calculation model that have later been corrected by other authors [9], the proposed description (and scoring system) for the progress of human decomposition is still the gold standard in this field.
The assessment was performed by at least two independent observers for the head/neck region, the trunk and the limbs, following the standardized scoring criteria.
All scores were reviewed and eventual variations in the documented assessments discussed until a consensus was reached. Additional morphological observations not included in the TBS system, such as local discolorations, desiccation, or insect activity, were documented with a combination of digital photos and detailed written notes.
To relate the observed morphological changes to PMI and temperature (and thereby test the applicability of existing prediction models), ADD were recorded during the outdoor exposure of the bodies at the research facility. Baseline ADD values for all donors were estimated with their TBS at the time of placement using the regression model by Moffatt et al. [9].
Protein analysis
Following a scalpel incision through the skin and underlying tissue, a 5 mm biopsy needle was used to collect in situ muscle samples. The muscle tissue (approximately 5 × 5 × 5 mm) was immediately transferred to vials containing 1.0 ml of RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor tablets (Roche) and stored in an on-site freezer until further processing. After each biopsy, the skin incision was sealed using cyanoacrylate adhesive and 3M^TM^Tegaderm™ film to prevent desiccation and potential entry points for insects or microbial contamination. Subsequent sampling was conducted every 24 h at a minimum distance of 1–2 cm from the previous sampling site.
Immediately after each in situ sampling, the extracorporeal tissue subsamples (approximately 5 × 5 × 5 mm) were collected from the excised tissue blocks using a scalpel, transferred to the same buffer system and stored under the same conditions.
For sample processing, all frozen muscle samples were homogenized using a standardized protocol [26]. Mechanical disruption was initially performed using an Ultra-Turrax homogenizer to disperse the tissue. This was followed by sonication using a VialTweeter (2 × 100 Ws per sample) to achieve further breakdown of cellular structures. The samples were then centrifuged at 1000 × g for 10 min, and supernatants were stored at −20 °C until further processing. Total protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific).
Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% resolving gels and 5% stacking gels. The resolving gel consisted of 10% acrylamide (acrylamide/bisacrylamide 37.5:1, 0.1% SDS, 0.05% TEMED, 0.05% APS, and 375 mM Tris–HCl, pH 8.8). The stacking gel was prepared with 5% acrylamide (acrylamide/bisacrylamide 37.5:1, 0.1% SDS, 0.125% TEMED, 0.075% APS, and 125 mM Tris–HCl, pH 6.8). Protein samples were adjusted to a final concentration of 30 μg (for vinculin, α-actinin, and GAPDH) or 10 µg (for tropomyosin) and mixed with SDS sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue, and 62.5 mM Tris–HCl, pH 6.75). The samples were denatured at 90 °C for 5 min and loaded onto the gels together with a prestained protein ladder (Roti Mark Tricolor, Carl Roth). Electrophoresis was run at 150 V for 2 h in running buffer containing 25 mM Tris, 195 mM glycine, 2 mM EDTA, and 0.1% SDS.
Following separation, proteins were transferred to polyvinylidene fluoride (PVDF) membranes using wet blotting. The PVDF membranes were activated with methanol and equilibrated in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). The protein transfer was then carried out for 75 min at 250 mA. Membranes were dried and stored at −20 °C until further use.
Membranes were blocked for 1 h in blocking buffer containing PBST (137 mM NaCl, 10 mM Na_2_HPO_4_ anhydrous, 2.7 mM KCl, 1.8 mM KH_2_PO_4_, 0.05% Tween 20) and 3% bovine serum albumin. The following monoclonal mouse primary antibodies were used for immunoblotting: anti-vinculin (1:1000, 7F9, Santa Cruz Biotechnology), anti-α-actinin (1:1000, H-2, Santa Cruz Biotechnology), anti-GAPDH (1:1500, 6C5, Santa Cruz Biotechnology), and anti-tropomyosin (1:500, CH1, DSHB). Polyclonal goat anti-mouse immunoglobulins conjugated with horseradish peroxidase (1:10000, Dako) were utilized as secondary antibodies. The incubation of the primary and secondary antibodies lasted for 1 h at room temperature in blocking buffer. Each antibody incubation was followed by washing steps (3 × 10 min) using PBST. Chemiluminescent signals were visualized using a luminol-based detection reagent (SuperSignal West Pico Plus, Thermo Scientific) and documented with a digital imaging system (iBright CL1000, Thermo Fisher Scientific).
Bands were quantified using ImageJ (v.1.54, National Institutes of Health, USA). Histogram-based analysis was used to calculate the intensity of each band, following the standard software protocol. The relative intensity was calculated in relation to the native band on day 1 (not day 0, as there were inconsistencies detected, likely due to partly frozen tissue at this time). Bands showing a relative intensity of ≥ 1% compared to the control were considered present, whereas intensities below this threshold were categorized as background.
Relative protein abundance was calculated for each time point and case. All analyses were performed using Microsoft Excel (v.16.98).
Results
Environmental conditions
Temperature, relative humidity and precipitation were continuously recorded throughout the two investigated periods. The ambient conditions exhibited minor differences between the two trials. In 2022, the mean air temperature during the investigated period was 22.7 °C (min. 16.2, max. 34.3 °C), while it was slightly cooler in 2024 with a mean of 19.8 °C (min. 13.8, max. 29.7 °C). Typical day-night fluctuations were observed during the initial five days in both trials, that became less distinct towards the end of the investigated periods (Fig. 1).Fig. 1. Temperature data during the investigation periods in 2022 and 2024. Ambient air temperatures were recorded at 15-min-intervals. Shown are ambient temperatures in 2022 (orange) and 2024 (dark blue), as well as temperatures within the extracorporeal storage box in 2024 (light blue)
Relative humidity was higher in 2024 with a mean of 84.0%, compared to 74.3% in 2022.
In 2022 a precipitation of less than 3 mm/day was recorded on four of seven days. In 2024, precipitation was observed on three days: two days exhibited moderate levels of 3.6 mm and 4.8 mm, while one intense precipitation event on day 6 reached 45.6 mm.
In addition to the ambient data, the temperature within the extracorporeal storage box was observed. In 2022 there was a technical malfunction and no data were recorded at all. After a similar failure on day 0 of the 2024 trial, the temperature inside the box was recorded from day 1 to 7 and showed a similar mean as ambient air temperature with 21.7 °C. However, daily fluctuations were more pronounced, ranging from 11.7 °C to 40.8 °C (Fig. 1).
Morphological assessment
It was observed that all donors exhibited a consistent morphological progression of decay over the 8-day observation period. However, the degree and rate of this progression varied between the two trials. In 2022, there were predominately signs of dry decomposition and early mummification observed, while the donors in 2024 exhibited mostly signs of moist decomposition. Overall, TBS values were higher in 2022 compared to 2024, reflecting a more advanced decomposition process (Fig. 2 a-b).Fig. 2. Morphological assessment of decomposition. (a) Representative morphological progression in body donors from the 2022 (top row) and 2024 (bottom row) trials at the day of placement (day 0), day 2, day 4 and day 7. (b) Temporal progression of mean total body scores (TBS) with standard deviations in 2022 (orange) and 2024 (blue) over the 8-day observation period. (c) Deviation (mean + SD) of the predicted ADD of the model from Moffatt et al. [9] to actually measured ADD across different ADD ranges in 2022 and 2024
At the time of placement (day 0), all donors exhibited signs consistent with early decomposition. In 2022, TBS values ranged from 5 to 9, with visible alterations such as mold formation on the head, skin slippage on the hands, gray-greenish discoloration of the trunk, and freezer burn artefacts.
On the second day, maggots were detected primarily in the facial and anogenital regions. By that time, a TBS of 11 had been reached. Between day 2 and day 3, decomposition advanced markedly reflected in a notable increase in TBS values to 15–17. At this stage, all donors exhibited epidermal slippage and increased maggot activity, accompanied by a change to brownish black discoloration and a leathery appearance of the trunk and limbs. From day 4 onwards, extensive maggot activity spread across the entire body surface as well as a remarkable leathery texture of the skin was detectable. By day 5, bone exposure was evident in two donors particularly in the facial and limb regions, accompanied early signs of mummification. These changes corresponded to TBS values ranging from 18 to 20, indicating a transition into an advanced decomposition stage. At the end of the observation period (day 7), TBS values ranged from 22 to 24 (Fig. 2 a-b).
In contrast, the 2024 donors were assessed with initial scores of 6 and 7, due to marbling and localized discoloration. One of the bodies also showed signs of freezer burn on the cheeks. On day 2, one donor displayed pronounced bloating accompanied by purging of decomposition fluid from the mouth, nose, eyes and ears. First maggot activity was observed on day 3, primarily around body orifices and became more extensive by day 5. During this period, TBS values increased from 11 and 12 to 13 and 14. Bone exposure in the head region was observed in one donor by day 6, corresponding to moist decomposition with less than half of the area skeletonized. On day 7, both donors exhibited bone exposure in the head region, while other body areas remained largely in the early stages of decomposition. The final TBS value was 16 in both donors, comparable to scores observed in 2022 on day 3–4 (Fig. 2 a-b).
To assess whether the observed decomposition progress in the present study was consistent with the improved equation of TBS and ADD by Moffatt et al. [9], the calculated ADD values for each TBS value were compared with the corresponding ADD estimates and associated confidence intervals reported in their study.
The prediction model performed accurately within the ADD 50–100 range in both years, showing only a 6% deviation (underestimation) from the observed values. For the ADD 100–200 range, the model slightly overestimated the accumulated degree days in 2022 (+ 10%) but substantially underestimated them in 2024 (−36%). Nevertheless, all deviations in these ranges were within the respectively proposed 95% confidence intervals. As the observation period in 2024 did not exceed ADD 200, no comparison was possible for higher values. In 2022, however, predictions for ADD > 200 were significantly overestimated by 51%, with two data points exceeding the 95% confidence interval limits (Fig. 2 c).
Muscle sampling
In both in situ and extracorporeal settings, sufficient muscle samples were obtained each day in both trials. In 2022 from day 4 onwards, the sampling site was affected by maggot activity and maggots were occasionally found in the biopsy needle (thus in the target muscle). A similar situation occurred in 2024, beginning on day 5. Despite these challenges, tissue collection continued as planned each day, and the sampled material could undoubtedly be identified as skeletal muscle.
In the extracorporeal setting, the muscle block was stored in a closed plastic container. In cases where donors were still partially frozen at the time of placement, substantial liquefaction was observed during thawing. Although tissue collection became slightly more challenging as decomposition progressed, it was effectively carried out according to the sampling protocol throughout the entire trial.
Protein degradation
All detected protein bands were clear and distinguishable, enabling reliable evaluation of protein stability over time. Native protein bands at placement (day 0) and day 1 were clearly detectable and largely in accordance with “fresh profiles” [16, 26, 27], despite the extended pre-placement intervals, thus providing a valid reference for subsequent degradation analyses. However, some of the samples collected on day 0 (especially when they were partially frozen) were later associated with reduced band intensity. Therefore, and to ensure comparability between samples, the native band detected on day 1 was used as a reference band.
The analyzed muscle proteins exhibited characteristic degradation pattern across both trials and sampling sources. Vinculin, α-actinin, and GAPDH depicted a breakdown into lower molecular weight fragments over time. In contrast, tropomyosin did not display any detectable degradation products.
A native vinculin band was detectable under all conditions at the beginning of the investigation period. In the 2022 samples, a distinct reduction of the relative band intensity occurred between day 1 and 3 and afterwards displayed a complete loss in most donors in both sample settings.
In contrast, the degradation process of the native band in the 2024 samples progressed at a slower rate. A more pronounced decline was observed in in situ samples after day 2, while the extracorporeal samples exhibited a continuous decrease over time. The standard deviations in this group suggest individual variation in the onset and rate of degradation (Fig. 3 e–f).Fig. 3. Postmortem degradation of vinculin in in situ and extracorporeal muscle tissue over an 8-day investigation (0–7 days) period in 2022 and 2024. (a-d) Representative Western blot images depicted the postmorten degradation pattern of the native vinculin band and the appearance of degradation products over time. (e–f) Relative band intensity of the native band of vinculin, normalized to day 1. Mean values and standard deviations are shown (2022: n = 3; 2024: n = 2)
In detail, the native vinculin band was no longer detectable between day 2 and day 3 in situ in all donors in 2022. In the extracorporeal samples, band loss was observed between day 2 and day 3 in two out of three donors. However, in one donor, the native band remained detectable until day 4. In 2024, the native vinculin band was consistently detectable in the in situ samples from one donor, whereas the other showed a complete (band) loss after day 3. In the extracorporeal setting, vinculin was present until day 6 in one donor and up to day 3 in the other (Fig. 3 a-d).
Among the identified degradation products, the 75 kDa and 63 kDa fragments were observed most consistently. In 2022, the 75 kDa fragment was detected in two of three donors in situ and in all donors in the extracorporeal setting. Although the time points of detectability varied considerably, the fragment was present at least until day 4 in all but one case. In 2024, it was observed in both donors under all conditions throughout the entire investigation period.
The 63 kDa fragment was present in all donors and sample settings in 2022, with variable onset and disappearance. In 2024, it was consistently detectable from day 2 onward, except in one extracorporeal sample, in which initial detection occurred on day 6.
Other fragments appeared sporadically and without consistent temporal patterns across donors, trials, or sampling settings.
In 2022, the native α-actinin disappeared between day 4 and 6 in both in situ and extracorporeal samples. In 2024, the native band remained detectable throughout the 8-day period in in situ samples in one donor, whereas in the other, the band could no longer be detected after day 4. In the extracorporeal samples, the native band was present until day 6 in one donor and was no longer detectable after day 2 in the other (Fig. 4 a-d). The evaluation of the mean relative abundance of α-actinin exhibited a progressive decline in both trials and sample settings. In 2022, the decline was more pronounced in both in situ and extracorporeal samples. In contrast, in 2024, the decrease was more gradual, with overall higher band intensities throughout the 8-day period (Fig. 4 e–f). The α-actinin degradation products, most notably at 70 kDa and 65 kDa lacked regularity in their appearance and degradation pattern, regardless of donors, trials or sample settings.Fig. 4. Postmortem degradation of α-actinin in in situ and extracorporeal muscle tissue over an 8-day investigation (0–7 days) period in 2022 and 2024. (a-d) Representative Western blot images depicted the postmorten degradation pattern of the native α-actinin band and the appearance of degradation products over time. (e–f) Relative band intensity of the native band of α-actinin, normalized to day 1. Mean values and standard deviations are shown (2022: n = 3; 2024: n = 2)
The degradation pattern of the native GAPDH band demonstrated some variations between trials, particularly in the in situ samples. In 2022, complete loss of the band in in situ samples occurred between day 3 and 4 in two of the three donors. In one donor, however, the band remained detectable until the end of the investigated period. In the extracorporeal setting, the band was detectable until day 4 or 5 in all donors. In 2024, the native GAPDH band remained detectable throughout the 8-day period in the in situ samples of both donors. In the extracorporeal setting, the band was present until day 6 in one donor, whereas the protein band of the other donor remained stable over the investigation period (Fig. 5 a-d). Analysis of the mean relative band intensities demonstrated a pronounced decline in 2022 in situ samples between day 1 and day 3, whereas the extracorporeal samples exhibited a more gradual decrease over the same period. In 2024, signal levels remained relatively stable, particularly in in situ samples, while a moderate decline was observed in the extracorporeal samples.Fig. 5. Postmortem degradation of GAPDH in in situ and extracorporeal muscle tissue over an 8-day investigation (0–7 days) period in 2022 and 2024. (a-d) Representative Western blot images depicted the postmorten degradation pattern of the native GAPDH band and the appearance of degradation products over time. (e–f) Relative band intensity of the native band of GAPDH, normalized to day 1. Mean values and standard deviations are shown (2022: n = 3; 2024: n = 2)
(Fig. 5 e–f). Degradation products of GAPDH were observed in both trials, predominantly within the first days of the investigation period. Bands at approximately 30, 28 and 26 kDa were detected across donors and sample settings with non-recurring temporal patterns.
Tropomyosin was consistently detectable as a native double band under all conditions throughout the investigated period in 2024. However, in the in situ samples, a decline in relative band intensity was detected over time. In the extracorporeal setting, the relative band abundance differed notably between donors (Fig. 6 e–f).Fig. 6. Postmortem degradation of tropomyosin in in situ and extracorporeal muscle tissue over an 8-day investigation (0–7 days)period in 2022 and 2024. (a-d) Representative Western blot images depicted the postmorten degradation pattern of the native double band of tropomoysin. (e–f) Relative band intensity of the native band of tropomyosin, normalized to day 1. Mean values and standard deviations are shown (2022: n = 3; 2024: n = 2)
In contrast, degradation of the native band was evident in 2022. The native double band was no longer detectable at specific time points in both in situ and extracorporeal samples. In the in situ samples, complete loss of the native bands was observed from day 4 or 6 in two of three donors. In the extracorporeal samples band loss occurred between day 4 and 5 in two donors. However, in one donor, no loss of the native bands was observed regardless of the sampling setting throughout the investigation period (Fig. 6 a-d).
The comparative overview of postmortem degradation in in situ muscle revealed a consistent temporal shift in the onset of the loss of the native protein band between the two study trials. In 2024, protein degradation occurred at later time points across all analyzed proteins compared to 2022. For instance, degradation of vinculin in 2024 ranged from day 4 to no detectable loss during the observation period, whereas in 2022, degradation occurred between day 2 and 3. Similarly, degradation of α-actinin in 2024 ranged from day 5 to no detectable loss. In contrast, 2022 samples showed a degradation of α-actinin between days 4 and 6. GAPDH and tropomyosin, which demonstrated a higher stability overall, remained detectable throughout the entire sampling interval in 2024. In 2022, degradation of GAPDH and tropomyosin was observed from day 3 and day 4, respectively, to no detectable loss during the investigation period depending on the donor (Fig. 7).Fig. 7. Comparison of the onset of complete degradation of native protein bands in in situ muscle samples over the 8-day investigation period in 2022 (n = 3) and 2024 (n = 2). Filled segments (blue or orange) indicate presence of the native band in all donors. Hatched segments (blue or orange) depict band loss in at least one donor and presence in at least another, therefore a transition period. Blank segments indicate absence of the native band in all donors
These findings highlight protein-specific differences in postmortem stability, with vinculin and α-actinin exhibiting accelerated degradation over GAPDH and tropomyosin. However, in donor-specific cases in which no loss of the native band was observed within the 8-day investigation period, the onset of degradation remained unclear.
To compare in situ and extracorporeal samples, the protein-specific degradation was assessed individually for each donor. For each protein, the onset of degradation was determined in both sample settings, and the donor-specific differences were compared. Protein degradation began on the same day in both sample settings in 33% of the evaluated cases. In 67% of the cases the onset differed no longer than one day, and in all cases the deviation did not exceed two days. The number of evaluable donors varied between proteins, as cases without detectable degradation were excluded. Across proteins, differences in degradation onset between in situ and extracorporeal samples ranged from 28 to 41% (Table 2). While the onset of degradation often varied depending on the protein and donor no consistent trend could be identified regarding temporal predominance of any of the settings.Table 2. Variability in the onset of protein degradation in in situ and extracorporeal samples. The number of donors (n) in which degradation of the native band was observed in both settings is given. Variability of degradation onset across donors was calculated as the standard deviation (SD) in % of time between in situ and extracorporeal samplesVinculinα-actininGAPDHTropomyosinn442**2SD*33%**28%**29%*41%
Discussion
The present study investigated postmortem decomposition under natural environmental conditions, comparing two study trials in 2022 and 2024 and examined protein degradation in both in situ and extracorporeal settings. The findings highlight the relevance of environmental factors on decomposition processes and outline the potential of an extracorporeal model for standardized investigation of extrinsic influences.
In both trials, all body donors exhibited progressive morphological changes over time, as reflected by increasing TBS values. However, the degree, temporal dynamics and distribution of decomposition varied considerably between 2022 and 2024.
Selected proteins exhibited temporally distinct degradation patterns across donors and environmental conditions, confirming their suitability as molecular markers for PMI estimation. The reproducibility of these patterns under variable field conditions emphasizes their forensic relevance. The dataset provides valuable reference data for protein-based PMI estimation, particularly within the intermediate postmortem interval where existing forensic methods are limited.
In 2022 decomposition advanced faster, as clearly reflected by consistently higher TBS values throughout the investigation period. All donors exhibited distinct desiccation and early signs of mummification, with bone exposure observed in the facial region and limbs. The final TBS values reached up to 24, corresponding to an advanced stage of decomposition across all body regions [8]. When predicting ADD using Moffatt et al.’s [9] model and comparing them to the measured values the model predicted morphological progression with good accuracy at low to moderate ADD levels, while higher ADD were increasingly overestimated. This pattern may indicate that the model overemphasized temperature-driven progression once decomposition has advanced.
In contrast decomposition in 2024 progressed more slowly and manifested differentially according to body region. The observed pattern was characterized by moist tissue conditions, delayed insect activity and only localized bone exposure, largely limited to the head region. The trunk and limbs remained in earlier stages of degradation throughout the observation period. Accordingly, TBS values did not exceed a value of 16, indicating region-specific decomposition patterns, with advanced decomposition restricted to the head. Moffatt et al.’s [9] model again performed well at low ADD but increasingly underestimated progression within the mid-range, while remaining within acceptable confidence intervals.
These differences in decomposition patterns generally align with the respective environmental parameters monitored during the two trials, particularly with respect to ambient temperature, relative humidity and precipitation. Both trials took place during the summer months, with moderate temperatures and thus active decomposition processes [19, 28, 29]. In 2022, average and maximum ambient temperatures were higher and relative humidity was distinctly lower than in 2024. These environmental parameters led to drier conditions, promoting desiccation and insect colonization, as reported in literature [29, 30].
In comparison, decomposition in 2024 occurred at a lower average temperature and higher relative humidity levels, resulting in elevated moisture retention. The presence of moisture rather facilitates microbial and enzymatic activity, promoting putrefaction [29]. To summarize, the environmental conditions in 2024 were favorable for microbial and enzymatic decomposition, whereas insect colonization was noticeably delayed compared to the trial in 2022. As these and other aspects are recognized as potential influencing factors for degradation, there are already efforts undertaken to address them to improve ADD calculation models [31, 32].
Previous studies have furthermore reported different effects of rainfall on insect colonization. While moderate to heavy rain was shown to inhibit flight activity and carcass colonization, other studies observed no significant reduction in oviposition [33–35]. In this study, only the 2024 field trial included a single heavier rainfall event on day 6, when insect activity had already been documented. However, colonization started by day 3, and reached a maximum by day 5, thus rainfall was not temporally aligned with the initial colonization phase and had no relevant impact. In addition to environmental factors, postmortem tissue conditions may have influenced insect colonization patterns. Some of the donors exhibited signs of incomplete thawing upon placement, with partial freezing of the tissue. Freezing has been shown to delay insect colonization, and in addition can damage tissue structures and alter gut microbiota, thereby influencing additional aspects of the decomposition process [28, 36].
Beyond observation of morphological differences, an analysis of muscle samples was performed to investigate protein degradation patterns. In contrast to the in situ setup, extracorporeal muscle tissue was stored in a plastic container placed inside a lightproof box under semi-controlled conditions. Mean temperatures inside the box were comparable to the ambient temperature but exhibited more pronounced day-night fluctuations. This setup generally allowed a more isolated investigation of temperature-dependent autolytic-proteolytic degradation (no insects and gut microbiota involved). However, in 2024, the presence of partially frozen tissue resulted in increased water accumulation within the containers, likely affecting the water content of the samples and thus diluting the overall amount of protein per sample.
Beyond the observed morphological difference an analysis of in situ muscle samples was performed to investigate protein degradation patterns. Despite the long pre-placement intervals and freezing conditions, the protein patterns at placement (day 0) and day 1 were largely consistent with those reported for fresh cases, showing no indication of advanced degradation [16, 26, 27]. This observation is supported by findings from a porcine model, demonstrating that protein degradation proceeds in predictable patterns that are largely unaffected by a freeze–thaw process [37]. Although these findings suggest that prolonged freezing does not substantially affect postmortem protein degradation, a potential effect cannot be fully excluded. The analysis revealed protein-specific differences in postmortem degradation susceptibility. Among the analyzed markers, vinculin and α-actinin were more susceptible to proteolytic degradation, as indicated by the relatively early loss of the native band and the appearance of characteristic degradation products. In contrast GAPDH and tropomyosin exhibited a comparatively higher stability, with the native band remaining detectable for extended periods in most cases. This observation aligns with previous studies, in which the native bands of GAPDH and tropomyosin remained detectable for at least 10 days postmortem [3, 38]. The different degradation patterns are consistent with previous studies and underscore the relevance of selecting proteins with varying susceptibility to degradation to characterize decomposition progress across different postmortem phases [3, 15, 17, 38, 39].
Despite the observed degradation products largely appeared at comparable molecular weights to those reported in earlier studies, their occurrence lacked temporal consistency across body donors and no regular pattern in their occurrence [17, 21, 38].
In 2022, all proteins depicted degradation patterns in earlier PMI stages, as well as an earlier insect colonization, consistent with the warmer, drier conditions. In contrast, in 2024, protein degradation progressed at a lower rate and the onset of insect activity was delayed. GAPDH and tropomyosin, in particular, remained detectable throughout the investigated sampling period. These patterns underscore the critical influence of environmental conditions on protein degradation. Consistent with previous studies, elevated temperatures were associated with accelerated proteolysis [26, 40, 41]. Higher relative humidity in 2024 may have contributed to the delayed protein degradation. Oh et al. [42] demonstrated in a rat model that autolysis progressed more gradually under humid conditions. High humidity reduces cellular desiccation and drought-induced stress, conditions that promote the release and activity of endogenous proteolytic enzymes [42].
While individual variation in the onset of native band loss was observed in both trials, donor-specific influences appear to have a relatively unimportant impact, compared to environmental factors. The present findings thus suggest that extrinsic factors have a stronger impact on postmortem degradation than intrinsic characteristics such as age, body mass or medical history, consistent with previous studies reporting the dominant role of environmental conditions in the decomposition process [6, 17, 38].
With the comparative extracorporeal degradation model, we confirmed findings from previous studies, showing that postmortem protein degradation is not limited to in situ decomposition but proceeds in a comparable and reproducible fashion in excised body parts [21, 26]. Proteins exhibited degradation patterns consistent with those observed in the in situ samples, including the protein-specific differences to proteolytic susceptibility. Notably, in one third of all evaluated cases, native band loss was observed on the exact same day in both in situ and extracorporeal samples, and in two thirds of the cases, the difference was no longer than a day. No consistent trend was observed across experimental conditions regarding an earlier, or later onset of degradation. These findings highlight that degradation onset was comparable between both sampling settings, which is further supported by the moderate variability observed across donors. Although temperature is considered the main influencing factor in postmortem proteolysis, additional factors, such as tissue hydration within the container may influence the timing of degradation [19, 42]. In contrast, in situ decomposition is clearly affected by additional environmental influences such as insect activity. The secretory and excretory proteases released by insect larvae may promote muscle protein degradation [12, 20]. In addition, maggot activity can induce higher local temperatures and thus further promote protein degradation [43, 44].
Such influences may also account for deviations in the onset of protein degradation between in situ and extracorporeal settings. However, the exact impact of insect activity on protein degradation remains to be investigated. Nevertheless, the extracorporeal model appears to be a promising model system to investigate isolated extrinsic decomposition factors under standardized conditions in human tissue. Although the experimental setting in the field was still subject to fluctuations, the model could well be adapted for the use under controlled laboratory conditions, enabling the investigation of defined parameters such as temperature, or insect activity.
Conclusion
Despite the small sample size and different environmental and individual influencing factors, this study provides a first comparative assessment of human postmortem decomposition under natural conditions and highlights the potential of extracorporeal human tissue model to investigate extrinsic influencing factors under controlled conditions. The observed differences in both morphological and molecular parameters between 2022 and 2024 emphasize the critical role of environmental factors, particularly temperature, relative humidity and insect activity.
While protein degradation patterns were reproducible across in situ and extracorporeal settings, inconsistencies in the onset of protein degradation require additional research. It must be aimed to bridge from detected band patterns to PMI under varying temperature regimes. Therefore, reliable prediction models (including confidence intervals) are required. This can likely only be reached from large scale experiments on multiple model systems, including autopsy cases (for “healthy”, minimal impacted human tissue) and extracorporeal models (for longitudinal studies under controlled conditions). However, it should be noted that the opportunities to study human decomposition under outdoor conditions also represent invaluable reference data for the development of more reliable protein-based PMI estimation methods and for enhancing our understanding of human decomposition dynamics within a forensic context.
The reference list from the paper itself. Each links out to its DOI / PubMed record.