Effect of temporary freezing on postmortem protein degradation patterns

Experimental design and sampling procedure

Six commercial sub-adult crossbreed pigs (German Large White × German Landrace, 4 months old, 50 ± 4 kg, 4 male, 2 female) were used for the experiment. To minimize possible bias from factory farming practices, experimental animals fed on quality feed from local production were obtained from controlled species-appropriate husbandry. Animals were killed in a certified slaughterhouse according to standard procedures by captive bolt stunning and subsequent exsanguination. Both hind limbs of each individual were separated by dissection with professional butcher cutlery immediately after death, giving a total of n = 12 limbs. A first set of samples from the M. biceps femoris (= 0 h reference samples) was taken from all hind limbs still in the slaughterhouse immediately after death, prior to transportation to the lab. For further processing, hind limbs were allocated to two treatment groups (n = 6) with different experimental setups.

The first group (referred to as “non-frozen”) was, without further delay, transferred to a climate chamber and incubated under constant conditions (temperature 30 ± 2 °C; humidity 50 ± 5% rH). Muscle samples were regularly collected at a total of 18 pre-defined time points after death: 0, 4, 8, 12, 16, 24, 32, 40, 48, 56, 64, 72, 80, 96, 112, 128, 144, 160 h postmortem (hpm).

The second group of hind limbs (referred to as “pre-frozen”) was first subjected to long-time storage in a deep-freeze room at constant − 20 °C for 4 months and then transferred and stored in the + 30 °C climate chamber under the same conditions applied to the non-frozen limbs. Sampling also followed the time schedule of the non-frozen limbs, although with two modifications: (i) The pre-frozen limbs were sampled in still frozen condition, directly after the transfer to the climate chamber (for technical modifications, see below). This provided the 0*haot baseline samples (haot = hours after onset of thawing), containing all marker proteins at onset-of-thawing state. (ii) Since full adaptation to + 30 °C took approximately 24 h (cf. Fig. 1), sampling of pre-frozen limbs was prolonged for another day. This enabled us to take two additional samples (at 168 and 184 haot, respectively) in order to ensure high comparability of the two experimental groups. Note: Sampling times of the pre-frozen group are expressed in hours after onset of thawing [haot]. However, in the interests of simplicity, in the present work, haot is often treated as equivalent to hpm.

Fig. 1figure 1

Temperature measurements throughout the first 75 h of the experiment until constant conditions were reached. Red lines indicate non-frozen rectal (thick line) and environmental (thin line) temperatures. Blue lines represent temperatures of frozen hind limbs. Temperature measurements of frozen limbs start with the onset of thawing/warming process (marked in transparent blue). Temperature data show that after approx. 12 h, the non-frozen hind limbs have cooled down to ambient temperature (marked in transparent red), whereas the frozen hind limb reached the equilibrium after approx. 24 h. After approx. 24 h, both treatment groups were at similar temperature conditions (Note: transient drops in environmental temperatures are due to opening of the climate chamber during sample collection.)

Sampling in both treatment groups was performed according to the following standardized procedure: An incision was made through the skin and the underlying fascial layer using a surgical scalpel, and muscle samples at a size of approx. 5 × 5 × 5 mm were excised from the M. biceps femoris at a depth of 2 cm within the belly of the muscle. A minimum distance of 2 cm was kept between successive sampling sites. Muscle samples were snap frozen and stored in liquid nitrogen until further processing. As the still-frozen condition of the limbs at 0*haot prevented scalpel incisions, these samples were obtained with the help of a power drill. After discarding skin and fatty tissue, borings of muscle tissue from a depth of 2 cm were collected and transferred to liquid nitrogen.

Temperature measurements

During both experimental time courses (non-frozen and pre-frozen treatment groups), environmental conditions in the climate chamber, as well as temperature data inside of one hind limb, as measured by a puncture sensor, were documented throughout the entire sampling time (cf. Fig. 1).

Temperature measurements of frozen limbs start with the onset of thawing/warming process, after a 4-month storage at − 20 °C. Temperatures of non-frozen hind limbs were documented directly after death and after being placed within the climate chamber. Temperature data show that after approx. 12 h, the non-frozen hind limbs have cooled down to ambient temperature, whereas the frozen hind took approx. 24 h. After approx. 24 h, both treatment groups were at similar temperature conditions.

Notably, during the placement of the hind limbs inside the chamber and setting up all the data loggers for temperature measurements, the chamber was open and the temperature dropped. It took approximately one and a half hours for the chamber to adjust the temperature again. Transient drops in environmental temperatures occurred during sample collection due to opening of the climate chamber.

Sample processing

Muscle samples were homogenized by cryogenic grinding and subsequent sonication with ultrasound (2 × 100 Ws/sample). A 10 × vol/wt RIPA buffer was used as lysis and extraction buffer, containing protease inhibitor cocktail (SIGMA) to prevent further protein degradation. Homogenized samples were centrifuged at 1.000 × g for 10 min, and supernatants transferred to separate tubes and stored at − 20 °C until further use. Total protein concentrations in the samples were measured using a Pierce BCA-Assay Kit (Thermo Fisher Scientific Inc.) and diluted to protein-specific values with double distilled water (30 µg for vinculin and alpha tubulin, 15 µg for alpha actinin, 10 µg for GAPDH).

SDS-PAGE and Western blotting

SDS-PAGE was performed according to the protocol of Laemmli [30] with some adaptions. Electrophoresis was run on 5% stacking gels (acrylamide/N,N′-bismethylene acrylamide = 37.5:1, 0.1% SDS, 0.125% TEMED, 0.075% APS, 125 mM Tris HCl, pH 6.8) and 10% polyacrylamide resolving gels (acrylamide/N,N′-bismethylene acrylamide = 37.5:1, 0.1% SDS, 0.05% TEMED, 0.05% APS, 375 mM Tris HCl, pH 8.8). The running buffer contained 25 mM Tris pH 8.3, 195 mM glycine, 2 mM EDTA, and 0.1% SDS. Samples diluted to adequate total protein content (10–30 µg) were denatured at 90 °C for 5 min prior to insertion into the stacking gel wells (volume 20 µl). Electrophoresis was performed at a constant voltage of 150 V until the dye front reached the bottom of the resolving gel (duration approximately 2 h). Following electrophoresis, proteins were transferred from the gels onto polyvinylidene fluoride (PVDF) membranes in transfer buffer containing 192 mM glycine, 20% methanol, and 25 mM Tris pH 8.3. Electroblotting was run at a constant current of 250 mA for 75 min. Membranes were then stored at − 20 °C until further use. For Western blotting, membranes were blocked for 1 h in a blocking buffer containing PBST (137 mM NaCl, 10 mM Na2HPO4 anhydrous, 2.7 mM KCl, 1.8 mM KH2PO4, 0.05% Tween) including 1% bovine serum albumin (BSA; albumin bovine fraction V, pH 7.0) and then for 1 h incubated with the following primary antisera: mouse-clonal anti-vinculin (7F9, Santa Cruz Biotechnology, 1:1000), mouse monoclonal anti-α-actinin (H-2, Santa Cruz Biotechnology, 1:1000), mouse monoclonal anti-α-tubulin (12G10, DSHB, 1:500), and mouse monoclonal anti-GAPDH (6C5, Santa Cruz Biotechnology, 1:1500). HRP-conjugated polyclonal goat anti-mouse immunoglobulin (Dako, 1:10,000) was applied as secondary antibody. All antibodies applied were diluted in blocking buffer. After each antibody application, membranes were extensively washed and rinsed in PBST (3 × 10 min). HRP-mediated specific antibody binding was visualized with chemiluminescence substrate (Roti®-Lumin plus, Carl Roth) and photographed using an iBright CL1000 Imaging System (Thermo Fisher Scientific).

Data interpretation and statistics

Band intensity of all proteins was measured using the gel analysis tool of the ImageJ software (v.1.48 NIH, National Institutes of Health, USA). Histograms of the tonal distribution of the images were plotted and the areas underneath the graphs were measured according to the program’s standard protocol. Band patterns of the 0-hpm samples were considered the native form of the protein and used as a control in both experimental series. All signals with ≥ 1% relative density (compared to the respective dominant control band) were considered a present protein band; all signals < 1% of the respective control band were considered background. This enabled binarization of the results and provided obtaining binary information of the absence (0) or presence (1) of proteins and their degradation products.

The abundance of bands per time point and the respective PMI [hpm] were then statistically analyzed and logistic regressions were calculated for all significant correlations of protein changes with a significance level above 0.95. This allows to predict the PMI at which the presence of a specific degradation product can be expected in a significant number of cases (confidence threshold = 95%) or at which time point the native protein (or splice variant) is completely degraded. The method also provides indication about when a change is more likely to have occurred than not (using P = 50% as a threshold). In addition, bivariate correlations between the chronology of protein degradation events and the PMI were calculated using Spearman’s rank correlation coefficient (Spearman’s ρ). For all proteins that gave a full set of data, i.e., those exhibiting distinct degradation events in all investigated hind limbs within the investigated time period of 160 hpm, an analysis of variance (ANOVA) was performed to evaluate possible differences between treatment groups. Since certain proteins showed no degradation in some of the hind limbs, their data had to be excluded from ANOVA evaluation since this specific data interpretation requires a “last time point an individual protein was present”. By simply using the last sampling point, it would most likely not represent the actual outcome because the protein band might be present long after the investigated time. Statistical analyses were performed using the SPSS Statistics 26 software (IBM, USA), MS Excel 2016, and RStudio (PBC).

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