Postmortem Muscle Research Articles

Are animal models predictive for human postmortem muscle protein degradation?

A most precise determination of the postmortem interval (PMI) is a crucial aspect in forensic casework. Although there are diverse approaches available to date, the high heterogeneity of cases together with the respective postmortal changes often limit the validity and sufficiency of many methods. Recently, a novel approach for time since death estimation by the analysis of postmortal changes of muscle proteins was proposed. It is however necessary to improve the reliability and accuracy, especially by analysis of possible influencing factors on protein degradation. This is ideally investigated on standardized animal models that, however, require legitimization by a comparison of human and animal tissue, and in this specific case of protein degradation profiles. Only if protein degradation events occur in comparable fashion within different species, respective findings can sufficiently be transferred from the animal model to application in humans. Therefor samples from two frequently used animal models (mouse and pig), as well as forensic cases with representative protein profiles of highly differing PMIs were analyzed. Despite physical and physiological differences between species, western blot analysis revealed similar patterns in most of the investigated proteins. Even most degradation events occurred in comparable fashion. In some other aspects, however, human and animal profiles depicted distinct differences. The results of this experimental series clearly indicate the huge importance of comparative studies, whenever animal models are considered. Although animal models could be shown to reflect the basic principles of protein degradation processes in humans, we also gained insight in the difficulties and limitations of the applicability of the developed methodology in different mammalian species regarding protein specificity and methodic functionality.

Comparisons of meat quality and muscle fibre characteristics on multiple pig breeds and sexes using principal component analysis

Comprehensive understanding of the effect of pig breed and sex on meat quality and post-mortem muscle conditions will be necessary to estimate meat quality post mortem. In this study, we applied multiple regression and principal component analysis (PCA) using conventional meat quality traits and muscle fibre characteristics on six pig breeds and different sexes. A total of 1374 pigs from six breeds [Berkshire, Duroc, Landrace, Meishan, Yorkshire, and LYD (Landrace × Yorkshire sows with Duroc as terminal sires) crossbreed] belonging to three sexes (male, castrated male and female) were used in this study. Most of the muscle fibre characteristics showed moderate to high significant correlations with conventional meat quality traits. In particularly, the cross-sectional area (CSAF) of muscle fibres was strongly correlated with muscle pH values, water-holding capacity values and lightness (0.10 < |r| <0.26 and P < 0.01). Multiple regression analysis by general linear model revealed that the effects of breed were significant on muscle pH24h, lightness, drip loss, and marbling score (P < 0.01), whereas sex had no significant effect on meat quality. In the muscle fibre characteristics, the effects of breed and sex were significant on composition of area and density of muscle fibres (P < 0.05). We observed that the Meishan breed was clearly inferior to other breeds, whereas the LYD or Berkshire breeds showed better meat quality and muscle fibre properties. Moreover, we observed clear separations between pig breeds based on the PCA, whereas the sexes in each breed presented a continuum cluster. In summary, we suggest that muscle pH24h, lightness, and drip loss are conventional meat quality traits that can be considered as quantitative factors important in estimating ultimate meat quality of pigs along with the CSAF of muscle fibres; we conclude that Meishan pigs have low meat quality, whereas Berkshire and LYD crossbred pigs have higher quality of pork.

Effects of dietary starch types on early postmortem muscle energy metabolism in finishing pigs

This study aimed to investigate the effects of different dietary starch types on early postmortem muscle energy metabolism in finishing pigs. Ninety barrows (68.0±2.0kg) were randomly allotted to three experimental diets with five replicates of six pigs, containing pure waxy maize starch (WMS), nonwaxy maize starch (NMS), and pea starch (PS) (amylose/amylopectin were 0.07, 0.19 and 0.28 respectively). Compared with the WMS diet, pigs fed the PS diet exhibited greater creatine kinase activity, higher adenosine triphosphate and adenosine diphosphate contents, lower phosphocreatine (PCr), adenosine monophosphate and glycogen contents, and lower glycolytic potential (P<0.05). Moreover, the PS diet led to reduced percentage of bound hexokinase activity, decreased level of phosphorylated AKT (P<0.05) and increased level of hypoxia-inducible factor-1α (P<0.05). In conclusion, diet with high amylose content might promote PCr degradation and inhibit the rate of glycolysis, followed by attenuation of early postmortem glycolysis in finishing pigs.

Effects of dietary energy sources on early postmortem muscle metabolism of finishing pigs.

ObjectiveThis study investigated the effects of different dietary energy sources on early postmortem muscle metabolism of finishing pigs.MethodsSeventy-two barrow (Duroc×Landrace×Yorkshire, DLY) pigs (65.0±2.0 kg) were allotted to three iso-energetic and iso-nitrogenous diets: A (44.1% starch, 5.9% crude fat, and 12.6% neutral detergent fibre [NDF]), B (37.6% starch, 9.5% crude fat, and 15.4% NDF) or C (30.9% starch, 14.3% crude fat, and 17.8% NDF). After the duration of 28-day feeding experiment, 24 pigs (eight per treatment) were slaughtered and the M. longissimus lumborum (LL) samples at 45 min postmortem were collected.ResultsCompared with diet A, diet C resulted in greater adenosine triphosphate and decreased phosphocreatine (PCr) concentrations, greater activity of creatine kinase and reduced percentage bound activities of hexokinase (HK), and pyruvate kinase (PK) in LL muscles (p<0.05). Moreover, diet C decreased the phosphor-AKT level and increased the hydroxy-hypoxia-inducible factor-1α (HIF-1α) level, as well as decreased the bound protein expressions of HK II, PKM2, and lactate dehydrogenase A (p<0.05).ConclusionDiet C with the lowest level of starch and the highest levels of fat and NDF could enhance the PCr utilization and attenuate glycolysis early postmortem in LL muscle of finishing pigs.

A mitochondrial protein increases glycolytic flux

The purpose of this study was to determine the role of mitochondria in postmortem muscle metabolism. Isolated mitochondria were incorporated into a reaction buffer that mimics postmortem glycolysis with or without mitochondrial electron transport chain inhibitors. Addition of mitochondria lowered pH values at 240 and 1440min regardless of inhibitors. Reduction in pH was accompanied by enhanced glycogen degradation and lactate accumulation. To explore the mechanism responsible for this exaggerated metabolism, mitochondrial preparations were mechanically disrupted and centrifuged. Resulting supernatants and pellets each were added to the in vitro model. Mitochondrial supernatants produced similar effects as those including intact mitochondria. To narrow further our target of investigation, mitochondrial supernatants were deproteinized with perchloric acid. The effect of mitochondrial supernatant was lost after perchloric acid treatment. These data indicate that a mitochondrial-based protein is capable of increasing glycolytic flux in an in vitro model and may partially explain acid meat development in highly oxidative AMPKγ3R200Q mutated pigs.

Phosphorylation inhibits the activity of μ-calpain at different incubation temperatures and Ca2+ concentrations in vitro.

This study aimed to investigate the effects of phosphorylation on the activity of μ-calpain and its sensitivity to temperature and Ca2+. For temperature sensitivity analysis, sarcoplasmic protein was treated with alkaline phosphatase (AP) and phosphatase inhibitor (PI) at 4, 25 and 37°C. The results showed that the degradation degree of μ-calpain in the AP group was significantly higher after incubation for 12h. For calcium sensitivity analysis, samples treated with AP and PI were incubated at 0.01, 0.05, 0.1 and 1mM Ca2+. The results showed that the degradation rate of μ-calpain was maximum in the AP group and minimum in the PI group at 0.01, 0.05 and 0.1mM Ca2+. The differences between the three groups reduced as concentration increased. These data demonstrate that phosphorylation plays a negative role in regulating μ-calpain activity. This study clarifies the regulatory mechanism of μ-calpain activation in vitro and/or in postmortem muscle.

Pig cognitive bias affects the conversion of muscle into meat by antioxidant and autophagy mechanisms

Slaughter is a crucial step in the meat production chain that could induce psychological stress on each animal, resulting in a physiological response that can differ among individuals. The aim of this study was to investigate the relationship between an animal’s emotional state, the subsequent psychological stress at slaughter and the cellular damage as an effect. In all, 36 entire male pigs were reared at an experimental farm and a cognitive bias test was used to classify them into positive bias (PB) or negative bias (NB) groups depending on their decision-making capabilities. Half of the animals, slaughtered in the same batch, were used for a complete study of biomarkers of stress, including brain neurotransmitters and some muscle biomarkers of oxidative stress. After slaughter, specific brain areas were excised and the levels of catecholamines (noradrenaline (NA) and dopamine (DA)) and indoleamines (5-hydroxyindoleacetic acid and serotonin (5HT)) were analyzed. In addition, muscle proteasome activity (20S), antioxidant defence (total antioxidant activity (TAA)), oxidative damage (lipid peroxidation (LPO)) and autophagy biomarkers (Beclin-1, microtubule-associated protein I light chain 3 (LC3-I) and LC3-II) were monitored during early postmortem maturation (0 to 24 h). Compared with PB animals, NB pigs were more susceptible to stress, showing higher 5HT levels (P<0.01) in the hippocampus and lower DA (P<0.001) in the pre-frontal cortex. Furthermore, NB pigs had more intense proteolytic processes and triggered primary muscle cell survival mechanisms immediately after slaughter (0 h postmortem), thus showing higher TAA (P<0.001) and earlier proteasome activity (P<0.001) and autophagy (Beclin-1, P<0.05; LC3-II/LC3-I, P<0.001) than PB pigs, in order to counteract the induced increase in oxidative stress, that was significantly higher in the muscle of NB pigs at 0 h postmortem (LPO, P<0.001). Our study is the first to demonstrate that pig’s cognitive bias influences the animal’s susceptibility to stress and has important effects on the postmortem muscle metabolism, particularly on the cell antioxidant defences and the autophagy onset. These results expand the current knowledge regarding biomarkers of animal welfare and highlight the potential use of biomarkers of the proteasome, the autophagy (Beclin-1, LC3-II/LC3-I ratio) and the muscle antioxidant defence (TAA, LPO) for detection of peri-slaughter stress.

Exogenous and Endogenous Factors Influencing Color of Fresh Meat from Ungulates

Biochemistry of post-mortem muscle tissue is complex, and several factors affect the fresh meat color and color stability, both of which influence consumer acceptance. Therefore, improving meat color and meat color stability is of significant value to the meat industry and consumers. While extensive literature is available on the color and color stability of domestic ungulates, literature on wild ungulates is notably lacking. With an increasing global demand for meats from wild ungulates, it is critical to identify the knowledge gaps regarding their color and color stability. The objective of this paper is to overview the exogenous and endogenous factors influencing the color and color stability of fresh meats from domestic and wild ungulates. The literature highlighted that the pre- and post-harvest factors influencing meat color and meat color stability are interrelated and not mutually exclusive. Current research indicates that the effects of several of these factors are specific to species, breed, and muscle source. Novel ways to manipulate these factors using a biosystems approach should be explored to improve color attributes of fresh ungulate meats.

Histone acetyltransferase inhibitors antagonize AMP-activated protein kinase in postmortem glycolysis.

ObjectiveThe purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK) activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality.MethodsA total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR, a specific activator of AMPK), AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II) and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases). After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured.ResultsActivation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates.ConclusionProtein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.

Effects of protein phosphorylation on color stability of ground meat.

The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin.

Influence of post-mortem muscle glycogen content on the quality of beef during aging

Abstract Introduction: Glycolic changes which occur post-mortem have an impact on the physical and sensory features of beef, which in turn determine the successive processes and influence such beef quality traits as colour, tenderness, and cooling loss. The aim of this study was evaluation of the post-mortem changes in bovine meat during aging, quantitative analysis of glycogen and lactic acid, as well as examination of their impact on technological and sensory quality of selected muscles from Holstein-Friesian × Limousin breed carcasses. Material and Methods: The study included three muscles of different metabolic qualities and sarcomere length: m. semitendinosus, m. longissimus dorsi, and m. psoas major, collected from nine bull carcasses aged 24 ±2 months. Results: Significant correlations were found between the volume of cooling loss on individual days of aging and the pH value of muscle tissue, lactic acid and glycogen content, as well as beef lightness. However, no significant dependency between the volume of glycogen and the intensity of red and yellow colours was detected. Conclusion: The colorimetric analysis of glycogen and lactic acid can be an effective tool in predicting the quality of beef.

Underlying connections between the redox system imbalance, protein oxidation and impaired quality traits in pale, soft and exudative (PSE) poultry meat

The connections between the redox imbalance in post-mortem muscle, early protein oxidation and the onset of pale, soft and exudative (PSE) condition in chicken breast are studied. PSE was induced by incubation of post-mortem chicken carcasses at 37°C for 200min. PSE-induced muscle consistently had faster pH decline and lower pH at 200min (5.84 vs. 6.59) and 24h (5.69 vs. 5.96), higher L∗ (54.4 vs. 57.3), and lower texture and water holding capacity (WHC) than normal meat. The activities of catalase, glutathione peroxidase and superoxide dismutase were significantly lower in PSE-induced samples than in the normal counterparts. PSE was more susceptible to proteolysis and protein oxidation than normal meat during succeeding chilled storage with more intense tryptophan and thiols depletion, higher protein carbonylation and more intense formation of protein cross-links. We provide plausible explanations to support the role of protein oxidation in the impaired quality PSE chicken.

Alteration of fatty acid profile and nucleotide-related substances in post-mortem breast meat of α-lipoic acid-fed broiler chickens

The present study was conducted to determine the effects of α-lipoic acid supplementation on post-mortem changes in the fatty acid profile and concentrations of nucleotide-related substances, especially those of a taste-active compound, inosine 5ʹ-monophosphate, in chicken meat.Mixed-sex broiler chicks aged 14 d were divided into three groups of 16 birds each and were fed on diets supplemented with α-lipoic acid at levels of 0, 100 or 200 mg/kg for 4 weeks. Blood and breast muscle samples were taken at 42 d of age under the fed condition and then after fasting for 18 h. The breast muscle obtained from fasted chickens was subsequently refrigerated at 2°C for one and 3 d.α-Lipoic acid supplementation did not affect any plasma metabolite concentration independently of feeding condition, while a slight increase in plasma glucose concentration was shown with both administration levels of α-lipoic acid. In early post-mortem breast muscle under the fed condition, α-lipoic acid had no effect on concentrations of fatty acids or nucleotides of ATP, ADP, and AMP.In post-mortem breast tissues obtained from fasted chickens, total fatty acid concentrations were markedly increased by α-lipoic acid feeding at 200 mg/kg irrespective of length of refrigeration. This effect was dependent on stearic acid, oleic acid, linoleic acid and linolenic acid. However, among fatty acids, the only predominantly increased unsaturated fatty acid was oleic acid.Dietary supplementation with α-lipoic acid at 200 mg/kg increased the inosine 5ʹ-monophosphate concentration in breast meat and, in contrast, reduced the subsequent catabolites, inosine and xanthine, regardless of the length of refrigeration.Therefore, the present study suggests that α-lipoic acid administration altered the fatty acid profile and improved meat quality by increasing taste-active substances in the post-mortem meat obtained from fasted chickens.

Postmortem muscle protein degradation in humans as a tool for PMI delimitation

Forensic estimation of time since death relies on diverse approaches, including measurement and comparison of environmental and body core temperature and analysis of insect colonization on a dead body. However, most of the applied methods have practical limitations or provide insufficient results under certain circumstances. Thus, new methods that can easily be implemented into forensic routine work are required to deliver more and discrete information about the postmortem interval (PMI). Following a previous work on skeletal muscle degradation in the porcine model, we analyzed human postmortem skeletal muscle samples of 40 forensic cases by Western blotting and casein zymography. Our results demonstrate predictable protein degradation processes in human muscle that are distinctly associated with temperature and the PMI. We provide information on promising degradation markers for certain periods of time postmortem, which can be useful tools for time since death delimitation. In addition, we discuss external influencing factors such as age, body mass index, sex, and cause of death that need to be considered in future routine application of the method in humans.

Inosine-5'-monophosphate is a candidate agent to resolve rigor mortis of skeletal muscle.

The object of the present study was to reveal the action of inosine-5'-monophosphate (IMP) toward myofibrils in postmortem muscles. IMP solubilized isolated actomyosin within a narrow range of KCl concentration, 0.19-0.20 mol/L, because of the dissociation of actomyosin into actin and myosin, but it did not solubilize the proteins in myofibrils with 0.2 mol/L KCl. However, IMP could solubilize both proteins in myofibrils with 0.2 mol/L KCl in the presence of 1 m mol/L pyrophosphate or 1.0-3.3 m mol/L adenosine-5'-diphosphate (ADP). Thus, we presumed that pyrophosphate and ADP released thin filaments composed of actin, and thick filaments composed of myosin from restraints of myofibrils, and then both filaments were solubilized through the IMP-induced dissociation of actomyosin. Thus, we concluded that IMP is a candidate agent to resolve rigor mortis because of its ability to break the association between thick and thin filaments.

Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle

Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle.

Identification of different domains of calpain and calpastatin from chicken blood and their role in post-mortem aging of meat during holding at refrigeration temperatures

The aim of this study was to develop a simple, specific and rapid analytical method for accurate identification of calpain and calpastatin from chicken blood and muscle samples. The method is based on liquid–liquid extraction technique followed by casein Zymography detection. The target compounds were extracted from blood and meat samples by tris buffer, and purified and separated on anion exchange chromatography. It has been observed that buffer (pH 6.7) containing 50mM tris-base appears to be excellent extractant as activity of analytes was maximum for all samples. The concentrations of μ-, m-calpain and calpastatin detected in the extracts of blood, breast and thigh samples were 0.28–0.55, 1.91–2.05 and 1.38–1.52Unit/g, respectively. For robustness, the analytical method was applied to determine the activity of calpains (μ and m) in eighty postmortem muscle samples. It has been observed that μ-calpain activity in breast and thigh muscles declined very rapidly at 48h and 24h, respectively while activity of m-calpain remained stable. Shear force values were also declined with the increase of post-mortem aging showing the presence of ample tenderness of breast and thigh muscles. Finally, it is concluded that the method standardized for the detection of calpain and calpastatin has the potential to be applied to identify post-mortem aging of chicken meat samples.

A chemometrics approach to analyze volatile molecules released by post-mortem bovine fast-twitch muscles

ABSTRACTIt is well known that beef produces volatile molecules. In this work, the detection of volatiles released by post-mortem bovine fast-twitch muscles (Musculus longissimus dorsi and Musculus cutaneus trunci) was done using GC/MS–SPME (gas chromatography/mass spectrum–solid-phase microextraction). The releases of volatile molecules were modeled against three factors (rigor-mortis, animal age and oxidative capacity) using a chemometrics approach (experimental design and partial least squares regression). The GC/MS–SPME technique produced more than 30 reproducible chromatographic peaks, but only 13 were associated significantly with two factors (rigor-mortis and animal age). The volatile profile was composed mainly of alcohols, aldehydes and alkanes. The factor “animal age” was the main variable related to the release of volatile molecules. The results strongly suggest that the release of volatile molecules change according to post-mortem metabolism and the animal age.

Proteolytic resistance of actin but not of myosin heavy chain during processing of Italian PDO (protected designation of origin) dry-cured hams

Proteomics is the approach of choice to study the fate of specific proteins, and it is very useful in identifying quality molecular markers. A combination of immunochemical and mass spectrometry analysis was used to assess the occurrence of proteolytic changes of actin and myosin heavy chain (MHC) proteins in pig biceps femoris skeletal muscle during processing of three Italian PDO dry-cured hams. Early post-mortem muscle displayed low levels of actin and myosin fragments. In spite of a high proteolysis index and the presence of active cathepsin D until the final ripening phase, during dry-cured ham processing, very low actin proteolysis and no generation of fragments from α-skeletal muscle isoform were found, while the identified fragments derived mainly from the cardiac actin isoform. On the other hand, MHC showed a remarkable degradation of its catalytic head, generating a C-terminal 135-kDa fragment. Based on its ability to interact with actin in vitro, this MHC fragment might have a role in stabilisation of actin. In conclusion, these results suggest that maintenance of skeletal muscle α-actin could reflect limited dismantling of the sarcomeric structure and be a useful marker to monitor the events that result in the typical texture of dry-cured ham.

Pathology of eosinophilic fasciitis and its relation to polymyositis.

The anatomical substrate of eosinophilic fasciitis (EF) was studied in 15 muscle biopsy specimens of this disease, six of which included the dermis and subcutaneous tissue. As controls, 94 postmortem muscle specimens from patients dying of non-muscular diseases were used. Of these 94 specimens, 22 (23.4%) showed practically no deep fascia and 72 specimens showed a single dense bundle of collagen with no distinction between deep fascia and epimysium. The 15 specimens of EF showed thickening and inflammatory infiltration of varying degrees in the deep fascia, epimysium, perimysium, endomysium and also in muscle. We conclude that the anatomical substrate of EF is not confined to the deep fascia, but involves other structures including mysia and muscle itself. Most reported cases of EF in the literature do not even describe muscle. A comparative study of 15 biopsy specimens of polymyositis and dermatomyositis with those of EF revealed only quantitative differences in the histopathological changes of muscle and mysia, inflammatory infiltrate and eosinophilia. We suggest that the diseases are more closely related than previously recognized.