Postmortem Proteolysis Research Articles

Myofibril Fragmentation Index as an Immediate Postmortem Predictor of Buffalo Meat Tenderness

A study was undertaken to compare and correlate the myofibril fragmentation index (MFI) of fresh buffalo longissimus steaks with Warner-Bratzler shear force (WBSF) and the subjective measurement of overall tenderness to suggest the suitability of MFI as a predictor of cooked buffalo meat tenderness. The study revealed a significant positive correlation (P < 0.01) between MFI and WBSF from day 1 to 8. The mean correlation coefficients (r2) of overall tenderness with MFI and WBSF were 0.61 and 0.73, respectively. This indicates that MFI could be used as an objective method of evaluation of tenderness of buffalo meat as it is strongly correlated with the overall tenderness and WBSF. Therefore, MFI of raw aged buffalo longissimus steaks can be used as a predictor of cooked buffalo meat tenderness enabling selection of tender meat for retail sale and processing. Furthermore, the results imply that significant tenderization occurs during the first 24 h postmortem. Practical Applications Myofibril fragmentation index appears to be an effective tool in predicting meat tenderness early postmortem as it was found to correlate with both Warner-Bratzler shear force values and objective measures of tenderness of cooked meat. This index is a very useful indicator of meat tenderness, particularly for muscles not big enough to determine shear force or sensory tenderness. This can be of special promise to the meat industry or retailers in developing countries where there is a need for early postmortem measurements to assess meat quality as early as possible after slaughter. A significant amount of tenderization occurs during the first 24 h postmortem as is obvious from the results; the industry can equip themselves to further enhance this early postmortem proteolysis so that the consumer acceptance of the final product can be improved.

Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50–70 kDa), F2 (Mr:40–60 kDa) and F3 (Mr:10–15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.

Calpain-1 activity in bovine muscle is primarily influenced by temperature, not pH decline1

The objectives of this study were to 1) determine the conditions (temperature and pH) that exist in early postmortem muscle of normally chilled and delay chilled beef carcasses to provide a model for in vitro work and 2) determine the mechanism by which early postmortem temperature/pH conditions found in beef muscle influence the enzymes that regulate the aging process in vitro. For objective 1, 7 finished beef animals (HCW 385 ± 8 kg) were harvested with the right sides subjected to normal chilling (2.3°C) approximately 1.25 h postmortem and the left sides subjected to ambient temperature (delay chilling; 22.6°C) for an additional 4.75 h postmortem and then allowed to chill at 2.3°C. Delay chilled carcasses had a more rapid pH decline (P < 0.05) and a slower rate of carcass cooling (P < 0.05). No differences were observed between normally chilled and delay chilled samples for sarcomere length or postmortem proteolysis of troponin T (TnT; P > 0.10). Warner-Bratzler shear force (WBSF) was reduced in steaks from normally chilled carcasses at 14 d (P < 0.05), while results indicated a strong, positive correlation between 14-d WBSF and 3-h longissimus dorsi muscle (LM) temperature (r = 0.67, P < 0.01) as well as a strong, negative correlation between 14-d WBSF and 6-h LM pH (r = -0.65, P < 0.02). These results were used to design the methodology for objective 2, where isolated myofibrils were subjected to μ-calpain digestion at 4 or 22°C with either a fast or slow initial pH decline. As expected, digestions with a fast initial pH decline had lower pH values in the early time points of the incubation (P < 0.05). No differences were detected in μ-calpain activity or in the degradation of intact TnT between the fast and slow pH decline treatments (P > 0.10); however, warmer digestions resulted in a tendency for increased activation of μ-calpain (P < 0.10) and a significant reduction in intact TnT (P < 0.05). Additionally, a temperature × time interaction was revealed in μ-calpain activity and in the degradation of intact TnT (P < 0.05). Specifically, assayed calpain activity was lower at 0.17, 0.33, 1, and 3 h and greater at 72 h in warmer digestions, while intact TnT disappearance was greater as both time and digestion temperature increased. Meat aging and μ-calpain activity are influenced by both temperature and pH, but more research is necessary to fully realize their relationships.

Composition and quality characteristics of carcasses from pigs divergently selected for residual feed intake on high- or low-energy diets1

The objective was to determine the extent to which feeding low-energy, high-fiber (LEHF) and high-energy, low-fiber (HELF) diets impacts meat quality and carcass composition of pigs divergently selected for residual feed intake (RFI). Two experiments were conducted in the divergently selected Iowa State University RFI lines: Exp. 1 evaluated carcasses of generation (G) 8 pigs fed on commercial feeders; Exp. 2 evaluated composition, pork quality, sensory, and postmortem proteolysis of pigs fed on electronic single-space feeders in G 8 and 9. Pigs (N = 177) in Exp. 1 were randomly assigned a pen (mixed sex and line; N = 8). Groups (n = 3) of pigs were slaughtered at a mean BW of 121.5 kg. Pigs in Exp. 2 (G8: n = 158; G9: n = 157) were randomly assigned to 1 of 6 pens of each diet per G. Pigs from G8 were slaughtered at a mean BW of 122.5 kg and G9 at a mean of 128.4 kg. Data were analyzed using the mixed procedure of SAS. Fixed effects were line, diet, sex, and all appropriate interactions. Random effects were group, pen, litter, and sire and covariate of off-test BW. For Exp. 2, G was added as a fixed effect and sensory day was added as a random effect when applicable. In Exp. 1, carcasses from low RFI (LRFI) pigs were leaner and had less fat depth (P < 0.01). Carcasses from pigs fed the LEHF diet had a lighter HCW and greater estimated percent lean than pigs fed HELF diet (P < 0.01). In Exp. 2, LRFI pigs on the HELF diet had the greatest loin depth (P < 0.01). Chops from HRFI pigs had greater drip loss, color scores, lean tissue a*, and percent lipid and lesser percent moisture than LRFI ( P< 0.05). Chops from pigs on the LEHF diet had lesser muscle L* values and greater percent moisture than chops from pigs fed the HELF diet (P < 0.05). Chops from LRFI pigs were juicer than those from HRFI pigs (P < 0.05). Protein extracted at d 2 postmortem from LRFI pigs on the LEHF diet had a greater 38 kDa desmin degradation product than protein from LRFI pigs fed the HELF diet (P < 0.05). Day 5 postmortem extracted protein from HRFI pigs had greater 38 kDa desmin degradation product than LRFI (P = 0.05). Pigs fed LEHF (P < 0.01) had adipose with a greater iodine value than adipose from HELF fed pigs. Pork sensory quality from pigs differentially selected for residual feed intake was not influenced by energy content of the diet the pigs were fed.

A post-transcriptional mechanism regulates calpastatin expression in bovine skeletal muscle1

The objective of this study was to investigate whether single nucleotide polymorphisms (SNP) in the calpain 1 (CAPN1), calpain 3 (CAPN3) and calpastatin (CAST) genes, which have been shown to be associated with shear force and tenderness differences in the skeletal muscle of cattle, contribute to phenotypic variation in muscle tenderness by modulating the transcriptional activity of their respective gene. The mRNA expression of the calpain and CAST genes was assessed in the longissimus lumborum muscle (LLM) of cattle from two herds located in distinct production zones on the east (New South Wales, NSW) and west (Western Australia, WA) of Australia. The cattle in the herds were mainly Brahman cattle (Bos indicus) with smaller populations of Angus cattle (Bos taurus). There were 191 steers in the WA herd and 107 steers and 106 heifers in the NSW herd. These herds were established by choosing cattle from the diverse population which had different single nucleotide polymorphism (SNP) genotypes at the CAPN1, CAPN3 and CAST loci. Using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the transcriptional activities of the CAPN1 and the CAST genes, but not the CAPN3 gene, were found to differ between favorable, positively associated with tenderness, and unfavorable, negatively associated with tenderness, allelic variants of these genes. These findings suggest that the muscle shear force and consumer taste panel differences in tenderness explained by the CAPN1 and CAST gene markers are a consequence of alterations in their mRNA levels, which may ultimately influence the protein activity of these genes, thereby altering the rate and(or) the extent of postmortem proteolysis in skeletal muscle. Of particular importance were the significantly lower type II and type III CAST 5' splice variant mRNA levels that were detected in the LLM muscle of Brahman and Angus cattle with 2 favourable alleles of the CAST:c.2832A > G polymorphism. Moreover, a reduction in the abundance of an alternative polyadenylated variant of the CAST transcript, terminated at the proximal polyadenylation site, provides a unique insight into the potential involvement of a post-transcriptional regulatory mechanism which may influence protein expression levels in bovine skeletal muscle.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM—Anabolic implants and meat quality1

Anabolic implants are routinely used in the finishing phase of beef production to improve animal performance and feed efficiency. Implanting during the feedlot phase on average increases ADG 18%, feed intake 6%, feed efficiency 8%, carcass weight 5%, and ribeye area 4% compared with nonimplanted controls. Implants reduce the cost of beef production, which is important given current high feed costs and beef prices. In a 1996 review of 37 implant trials, the use of a combination (i.e., estrogenic and trenbolone acetate) implant increased returns by US$77/head compared with nonimplanted steers. If calculated using today's prices, a combination implant would increase returns by $163/head. However, concerns about potential negative effects of implants on marbling scores, quality grades, and tenderness exist. Changes in Warner-Bratzler shear force values of steaks from implanted steers are small (<0.5 kg) and appear related to an increase in initial tenderness, possibly due to hypertrophy of muscle fiber, instead of alterations in postmortem proteolysis. The increase in ribeye size observed with implanting may also reduce marbling scores through a dilution effect. The impact of anabolic implants on gene expression has shown that implanting downregulates expression of certain lipogenic genes (e.g., stearoyl-CoA desaturase, fatty acid synthetase, fatty acid elongase-6) in steers with low quality grades (Select-) but not in implanted steers with high quality grades (Choice-). Examination of the adipocyte's transcriptome has shown that 36 genes were differentially expressed due to implant treatment. More research is needed to further determine how anabolic implants alter lipogenic gene expression to address changes in marbling deposition with implant usage. Given our current high feed costs and cattle prices, anabolic implants are one of the most cost-effective technologies that can be used in beef production systems.

Comparison of postmortem proteolysis between Pekin and Muscovy duck breast muscles

Duck muscle is a popular source of red meat in Asia. However, information regarding the postmortem proteolysis of skeletal muscle between duck species is very limited. Therefore, the purpose of this study was to compare the postmortem calpain and desmin degradations between Pekin (PD) and Muscovy (MD) duck breast muscles stored at 5°C. The pH and μ/m-calpain activity were not different (P > 0.05) between PD and MD postmortem muscles. However, μ-calpain activity and desmin content decreased more rapidly (P < 0.05) in PD than in MD samples. Therefore, our results suggest that the postmortem proteolysis is more rapid and extensive in breast muscle from PD compared to MD.

Postmortem proteolysis in three muscles from growing and mature beef cattle

The objective of this study was to determine calpain system activity and postmortem protein degradation in three muscles from growing (n=6, 7.3±0.5months) and mature (n=6, 106.7±43.1months) beef cattle. The ratio of μ-calpain:total calpastatin activity tended to be lower in mature animals (P=0.08), suggesting reduced potential for proteolysis. Additionally, muscles from the mature group had greater calpastatin activity compared to calves at 6days postmortem and had less μ-calpain autolysis and troponin-T and titin degradation during the aging period (P<0.01). Between the longissimus, semimembranosus, and triceps brachii muscles, the triceps brachii had the least postmortem proteolysis, with greater calpastatin activity and less troponin-T and titin degradation compared to other muscles (P<0.01). These data suggest that calpastatin activity in muscle from older animals is more persistent postmortem. This difference may contribute to the decreased protein degradation and increased toughness of beef from mature cattle, even after aging.

Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein.

Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrPSc), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP (CtmPrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrPSc was demonstrated in the brains of recipient transgenic mice. This PrPSc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.

Caspase-3 does not enhance in vitro bovine myofibril degradation by µ-calpain1

Tenderness is a key component of palatability, which influences consumers' perception of meat quality. There are a variety of factors that contribute to the tenderness of beef carcasses, including postmortem proteolysis. A more complete understanding of this biological mechanism regulating tenderness is needed to ensure consistently tender beef. Numerous reports indicate µ-calpain is primarily responsible for the degradation of proteins postmortem. Meanwhile, it has been shown that caspase-3 can cleave calpastatin, the inhibitor of µ-calpain. Therefore, the objective of this study was to determine if in vitro degradation of calpastatin by caspase-3 can enhance the postmortem breakdown of myofibrillar proteins by µ-calpain. Bovine semitendinosus muscles were excised from two carcasses 20 min postmortem. Muscle strips were dissected from the semitendinosus, restrained to maintain length, and placed in a neutral buffer containing protease inhibitors. Upon rigor completion, myofibrils were isolated from each strip, and sarcomere length was determined. Samples with similar sarcomere lengths were selected to minimize the effect of sarcomere length on proteolysis. Myofibrils were then incubated at 22°C with either µ-calpain, µ-calpain+calpastatin, µ-calpain+caspase-3+calpastatin, or caspase-3+calpastatin for 0.25, 1, 3, 24, 48, or 72 h at a pH of 6.8. Proteolysis of troponin T (TnT) and calpastatin was evaluated using SDS-PAGE and Western blotting techniques. Analysis of Western blots confirmed significant degradation of calpastatin by caspase-3 (P<0.05). Additionally, Western blots revealed intact calpastatin disappeared rapidly as a result of digestion by µ-calpain. Although caspase-3 did not significantly degrade TnT (P>0.05), all µ-calpain digestion treatments resulted in substantial TnT breakdown (P<0.05). Degradation of TnT did not differ between the µ-calpain+calpastatin and µ-calpain+caspase-3+calpastatin digestions (P>0.05). Results of this study indicate caspase-3 cleavage of calpastatin does not enhance in vitro degradation of TnT by µ-calpain.

Significant role of μ-calpain (CANP1) in proliferation/survival of bovine skeletal muscle satellite cells

Calpains are a family of Ca2+-dependent intracellular cysteine proteases, including the ubiquitously expressed μ-calpain (CANP1) and m-calpain (CANP2). The CANP1 has been found to play a central role in postmortem proteolysis and meat tenderization. However, the physiological roles of CANP1 in cattle skeletal satellite cells remain unclear. In this study, three small interference RNA sequences (siRNAs) targeting CANP1 gene were designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Suppression of CANP1 in satellite cells was evaluated using these shRNA expressing constructs. Our results revealed that all three siRNAs could downregulate the expression of CANP1. Suppression of CANP1 significantly reduced cell viability in cell proliferation when compared with control cells. We found a crosstalk between CANP1 and caspase systems, particularly suppression of CANP1 resulted in an increase in the expressions of apoptotic caspases such as caspase-3, caspase-6, caspase-7, caspase-8, and caspase-9, as well as heat-shock protein (HSP) systems. Additionally, suppression of CANP1 led to the upregulation of other apoptosis and DNA damage-regulating genes whilst at the same time downregulating proliferation, migration, and differentiation-regulating genes. The results of our findings report for the first time that suppression of CANP1 resulted in the activation of caspase and HSP systems which might in turn regulate apoptosis through the caspase-dependent cell death pathway. This clearly demonstrates the key roles of CANP1 in regulation of cell proliferation and survival.Electronic supplementary materialThe online version of this article (doi:10.1007/s11626-013-9666-5) contains supplementary material, which is available to authorized users.

Evidence of decreased muscle protein turnover in gilts selected for low residual feed intake1

The objective of this study was to evaluate the contribution of muscle protein turnover (synthesis and degradation) to the biological basis for genetic differences in finisher pigs selected for residual feed intake (RFI). Residual feed intake is defined as the difference between expected feed intake (based on the achieved rate of BW gain and backfat depth of individual pigs) and the observed feed intake of the individual pig. We hypothesized that protein turnover would be reduced in pigs selected for low RFI. Twelve gilts from a line selected for 7 generations for low RFI and 12 from a contemporary line selected for 2 generations for high RFI were paired by age and BW and fed a standard corn-soybean diet for 6 wk. Pigs were euthanized, muscle and liver samples were collected, and insulin signaling, protein synthesis, and protein degradation proteins were analyzed for expression and activities. Muscle from low RFI pigs tended to have less μ- and m-calpain activities (P = 0.10 and 0.09, respectively) and had significantly greater calpastatin activity and a decreased μ-calpain:calpastatin activity ratio (P < 0.05). Muscle from low RFI pigs had less 20S proteasome activity compared with their high RFI counterparts (P < 0.05). No differences in insulin signaling intermediates and translation initiation signaling proteins [mammalian target of rapamycin (mTOR) pathway] were observed (P > 0.05). Postmortem proteolysis was determined in the LM from the eighth generation of the low RFI pigs versus their high RFI counterparts (n = 9 per line). Autolysis of μ-calpain was decreased in the low RFI pigs and less troponin-T degradation product was observed at 3 d postmortem (P < 0.05), indicating slowed postmortem proteolysis during aging in the low RFI pigs. These data provide significant evidence that less protein degradation occurs in pigs selected for reduced RFI, and this may account for a significant portion of the increased efficiency observed in these animals.

Evaluation of feedlot cattle working chute behavior relative to temperament, tenderness, and postmortem proteolysis

The objective was to investigate if the association between working chute behavior and beef tenderness found in our previous study is related to protein degradation and calpain system activity. Crossbred steers (n=183) allotted to 16 pens were weighed every 28d. Temperament was evaluated as exit velocity (EV), chute score (CS), and catch score (CAPS). Between 14 and 16mo of age (606±52kg), steers were harvested. Strip steaks were collected and aged for 14d. Subsamples were collected at 36h and 7d postmortem and analyzed for calpastatin activity, μ-calpain autolysis, and troponin-T degradation. Shear force (WBSF) was correlated (P<0.05) with calpastatin activity and measurements of troponin-T. Calpastatin activity, μ-calpain autolysis, and troponin-T measurements did not correlate with the measurements of EV, CS, and CAPS. Therefore, activation of the calpain system or differences in protein degradation did not appear to influence the differences in tenderness that are correlated with working chute behavior.

Proteolytic activities in fillets of selected underutilized Australian fish species

The hydrolytic activity of major endogenous proteases, responsible for proteolysis of myofibrillar proteins during post-mortem storage, may be an indicator of the textural quality of fish which influences consumer purchasing behaviour and thus market value of the final product. Furthermore, it may also influence the type and bioactive properties of the peptides released during post-mortem proteolysis of myofibrillar proteins. This study compared the activities of cathepsins B, B+L, D, H and calpain-like enzymes in crude muscle extracted from 16 Australian underutilized fish species. Fish species had a significant effect on the activity of these enzymes with barracouta showing the highest cathepsins B, B+L, D and H activities. Activities of cathepsins B and B+L were higher than cathepsin H for all studied species. The more commercially important rock ling and tiger flathead demonstrated higher cathepsin B+L activity, whereas gemfish and eastern school whiting showed higher activity towards cathepsin B. Underutilized fish species showing higher endogenous protease activities may be suitable for fish sauce production, whereas those with lower protease activities for surimi processing.

μ-Calpain is involved in the postmortem proteolysis of gizzard smooth muscle

Postmortem changes in proteins that have been implicated in affecting muscle integrity were examined in goose (GG) and duck (DG) gizzard smooth muscle stored at 5°C. GG and DG smooth muscles were sampled at 0, 1, 3 and 7day of storage. The pH was approximately 7 in both GG and DG samples during postmortem storage. Casein zymograms showed that 0-day μ-calpain activity was higher (p<0.05) in GG than in DG samples. As postmortem time progressed, μ-calpain was activated and autolyzed more extensively in GG than in DG samples. However, μ/m-calpain remained relatively stable in both samples. Western blots indicated that postmortem desmin degradation was more rapid in GG than in DG samples. In contrast, α-actinin remained nearly unchanged in both samples. Therefore, our results suggest that μ-calpain has an important role in the postmortem proteolysis of gizzard smooth muscle.

The Rates of Postmortem Proteolysis of Glutamate Transporters Differ Dramatically between Cells and between Transporter Subtypes

Glutamate transporters (GLT-1, GLAST, EAAC1) limit the actions of excitatory amino acids. Because a disturbed transporter operation can cause or aggravate neurological diseases, transporters are of considerable neuropathological interest. Human samples, however, are seldom obtained fresh. Here, we used mice brains to study how fast glutamate transporters are degraded after death. Immunoblots showed that terminal GLT-1 epitopes (within residues 1-26 and 518-573) had mostly disappeared after 24 hr. GLAST termini (1-25 and 522-543) degraded slightly slower. In contrast, epitopes within central parts of GLT-1 (493-508) and the EAAC1 C-terminus (510-523) were readily detectable after 72 hr. The decline in immunoreactivity of the GLT-1 and GLAST termini was also seen in tissue sections, but proteolysis did not happen synchronously in all cells. At 24 hr, scattered cells remained strongly immunopositive, while the majority of cells were completely immunonegative. GLAST and GLT-1 co-localized in neocortical tissue, but at 12 hr, many GLAST-positive cells had lost the GLT-1 termini. The uneven disappearance of labeling was not observed with the antibodies to GLT-1 residues 493-508. The immunoreactivity to this epitope correlated better with the reported glutamate uptake activity. Thus, postmortem delay may affect epitopes differently, possibly causing erroneous conclusions about relative expression levels.

Deficiency of Capn4 Gene Inhibits Nuclear Factor-κB (NF-κB) Protein Signaling/Inflammation and Reduces Remodeling after Myocardial Infarction

Calpain has been implicated in acute myocardial injury after myocardial infarction (MI). However, the causal relationship between calpain and post-MI myocardial remodeling has not been fully understood. This study examined whether deletion of Capn4, essential for calpain-1 and calpain-2 activities, reduces myocardial remodeling and dysfunction following MI, and if yes, whether these effects of Capn4 deletion are associated with NF-κB signaling and inflammatory responses in the MI heart. A novel mouse model with cardiomyocyte-specific deletion of Capn4 (Capn4-ko) was employed. MI was induced by left coronary artery ligation. Deficiency of Capn4 dramatically reduced the protein levels and activities of calpain-1 and calpain-2 in the Capn4-ko heart. In vivo cardiac function was relatively improved in Capn4-ko mice at 7 and 30 days after MI when compared with their wild-type littermates. Deletion of Capn4 reduced apoptosis, limited infarct expansion, prevented left ventricle dilation, and reduced mortality in Capn4-ko mice. Furthermore, cardiomyocyte cross-sectional areas and myocardial collagen deposition were significantly attenuated in Capn4-ko mice, which were accompanied by down-regulation of hypertrophic genes and profibrotic genes. These effects of Capn4 knock-out correlated with restoration of IκB protein and inhibition of NF-κB activation, leading to suppression of proinflammatory cytokine expression and inflammatory cell infiltration in the Capn4-ko heart after MI. In conclusion, deficiency of Capn4 reduces adverse myocardial remodeling and myocardial dysfunction after MI. These effects of Capn4 deletion may be mediated through prevention of IκB degradation and NF-κB activation, resulting in inhibition of inflammatory responses.

Changes in proteolytic enzymes mRNAs and proteins relevant for meat quality during myogenesis and hypoxia of primary bovine satellite cells

The current study was conducted to evaluate the functions of μ-calpain (CAPN1), calpastatin, HSPs (heat shock proteins), and caspases during myogenesis and cell death induced by sodium azide (NaN(3)) hypoxia. The cell samples were divided into three groups: satellite cells formed at confluent monolayer (stage 1), stage 1 cells fusion into myotubes on d eight post-differentiation (stage 2), and stage 2 cells treated with 1 mM NaN(3) for 24 h (stage 3). Real-time RT-PCR showed that stage 2 cells had increased CAPN1, calpastatin, caspase 7, and CARD9 (Caspase activation and recruitment domain 9) mRNA expressions compared to stage 1 cells (*p < 0.05). By Western blotting caspase 3, caspase 7, caspase 8, and caspase 9 protein levels increased in cells at stage 2 compared to cells at stage 1 (*p < 0.05). Real-time RT-PCR showed that stage 3 cells had increased CAPN1, calpastatin, caspase 7, HSP70 (70 kDA heat shock proteins), and HSP90 (90 kDA heat shock proteins-alpha) and decreased CARD9 mRNA expression compared to stage 2 cells (*p < 0.05). Stage 3 samples had increase caspase 7 and caspase 12 activities compared to stage 2 samples, and by Western blotting protein levels of both HSP70 and HSP90 expressions, increased significantly under hypoxia condition (*p < 0.05). Here, we conclude that CAPN1, calpastatin, caspase 3, caspase 7, caspase 8, and CARD9 have important roles for satellite cell myogenesis; and that caspase 7, 12, HSP70, and HSP90 are involved in the process of apoptotic cell death under hypoxia conditions and we speculate that these proteins may be involved in early postmortem proteolysis and meat tenderization.

Profile of biochemical traits influencing tenderness of muscles from the beef round

The objective of this study was to define the biochemical differences that govern tenderness and palatability of economically important muscles from the beef round using cuts with known tenderness differences. At 24h postmortem, the longissimus dorsi (LD), gracillus (GR), adductor (AD), semimembranosus (SM), sartorius (SAR), vastus lateralis (VL), and vastus intermedius (VI) muscles were removed from ten market weight beef cattle. Sensory and biochemical characteristics were determined in each cut and compared with the LD. The GR, SAR and VI had sensory traits similar to the LD while the SM, AD and VL differed. The GR, SAR, AD, and SM all had multiple biochemical characteristics similar to the LD, while the VI and AD had numerous biochemical differences. While no one biochemical characteristic can be used to predict tenderness across all muscles, analysis of the biochemical characteristics revealed that in most beef round cuts postmortem proteolysis provided a good indication of the tenderization occurring during aging.

Postmortem role of calpains in Pekin duck skeletal muscles

It is generally agreed that calpains are involved in postmortem proteolysis of skeletal muscle and improve meat tenderness. However, little information regarding the postmortem role of calpains in duck skeletal muscle is known. Therefore, the purpose of this study was to examine the role of calpains in Pekin duck postmortem breast muscles (BM) and leg and thigh muscles (LM) muscles at 5 °C. The postmortem pH was lower (P < 0.05) in BM than in LM. Western blots indicated that postmortem desmin degradation and the 30/32 kDa troponin-T degradation product accumulation were more rapid in BM than in LM. Casein zymograms showed that at-death µ-calpain activity was higher in BM than in LM. As time post mortem increased, µ-calpain was activated and autolyzed more rapidly and extensively in BM than in LM, but µ/m-calpain was activated at a relative slower rate compared with µ-calpain. Correlation results showed that µ-calpain activity, rather than µ/m-calpain activity, in BM samples was highly correlated with the abundance of desmin and the 30/32 kDa troponin-T degradation components across the postmortem period. However, no such correlations were found with LM µ- and µ/m-calpains. Therefore, our results suggest that BM µ-calpain with a faster and more extensive activation and autolysis would play a relatively dominant role in dictating degradation of desmin and troponin-T in postmortem duck muscle.