Ronald G. Bardsley

Tenderness - an enzymatic view.

One of the most common causes of unacceptability in meat quality is toughness. Toughness is attributed to a range of factors including the amount of intramuscular connective tissue, intramuscular fat, and the length of the sarcomere. However, it is apparent that the extent of proteolysis of key proteins within muscle fibres is significant determinant of ultimate tenderness. The objective of this manuscript is to describe the main endogenous proteolytic enzyme systems that have the potential to be involved in muscle post-mortem proteolysis and whether the experimental evidence available supports this involvement.

Effect of β-agonists on expression of calpain and calpastatin activity in skeletal muscle

Administration of beta-adrenergic agonists to domestic species can lead to skeletal muscle hypertrophy, probably by reducing the rate of myofibrillar protein breakdown. Myofibrillar breakdown is associated with the calcium-dependent proteinase system (calpains I,II and calpastatin) whose activity also changes during beta-agonist treatment. A number of growth trials using the agonists cimaterol and clenbuterol with cattle, sheep, chicken and rat are reported which suggest a general mechanism whereby beta-agonists reduce calpain I activity, but increase calpain II and calpastatin activity in skeletal muscle. Parallel changes in specific mRNAs indicate that changes in gene expression or stabilisation of mRNA could in part explain the changes in activity.

The relationship between slow and fast myosin heavy chain content, calpastatin and meat tenderness in different ovine skeletal muscles.

The present study investigated the relationship between fibre type distribution and slow (MHC-s) and fast (MHC-f) myosin heavy chain content on calpastatin and meat tenderness in longissimus dorsi (LD), tensor fasciae latae (TFL), semitendinosus (ST), trapezius (TZ) and supraspinatus (SS) muscles from six Mule×Charolais rams. Samples taken at slaughter were frozen either in liquid N(2) for analysis of MHC-s and MHC-f by immunoblotting, or in cooled isopentane for histochemical fibre typing. Calpastatin activity and an immunoreactive 135 kDa calpastatin band were analysed in samples taken 24 h postmortem. Shear force was determined on muscle chops taken at 24 h postmortem and conditioned until day 14. The intensity of MHC-s and MHC-f immunopositive bands correlated with %Type I and %Type II fibres identified histochemically (r(2)=0.612 and 0.366, respectively, p<0.001). Muscle specific differences were observed in MHC-s and MHC-f immunoreactivity, fibre type distribution, calpastatin activity, calpastatin 135 kDa immunoreactivity and shear force. MHC-s correlated positively with calpastatin activity (r(2)=0.725, p<0.001) and 135 kDa calpastatin (r(2)=0.228, p<0.01) across all muscle types. The data show that detection of MHC-s can be used to identify fibre type differences between ovine muscles and that this correlates with differences in calpastatin content and inhibitory activity, but not tenderness.

The effect of altered growth rates on the calpain proteolytic system and meat tenderness in cattle

The present study was conducted to determine the effects of growth pattern on the calpain system and meat tenderization. Twenty-four Friesian calves were randomly allocated to three treatment groups: FAST (fast growth rate), SLOW (severely restricted growth rate) and ALTER (restricted growth for 30 days followed by fast growth rate). Four animals from each group were slaughtered on day 32 or 45 after altering the growth rates. Samples of M. longissimus dorsi were rapidly frozen at slaughter for protein analysis by Western blotting. Restricted growth reduced the immunoreactivity of a calpastatin band (135 kDa) measured at 24 h postmortem. Immunoreactivity associated with the large subunit of μ- or m-calpain appeared to be unaffected by growth patterns. Shear force measurements taken after 14 days of conditioning were positively related to 135 kDa calpastatin at 24 h postmortem. In this study there was no clear relationship between shear force and growth pattern.

Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles.

The present study investigated the relationship between muscle type and components of the caspase protease system in porcine trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) muscles. Muscles were classified according to slow and fast myosin heavy chain (MHC) content determined by western blotting. MHC slow, but not MHC fast protein expression was significantly different between muscles (p<0.001). Protein levels of caspases 3, 8 and 12 and the caspase inhibitor apoptosis repressor with caspase recruitment domain (ARC) were determined. In addition the level of caspase 3 mRNA and activity levels of caspase 3/7 were determined. There was a significant difference in protein levels and activity between muscles (p<0.01), although no difference was observed in mRNA abundance. The data show that multiple components of the caspase system are expressed in porcine skeletal muscle and that their levels are variable, but there is not a distinct association of expression with a particular muscle.

Label free capacitive immunosensor for detecting calpastatin — A meat tenderness biomarker

An immunological capacitive biosensor for calpastatin was developed, optimized and applied for the analysis of meat extract samples. Anti-calpastatin antibody was immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid and Protein A from Staphylococcus aureus, and the obtained immunosensor was inserted as the working electrode in an electrochemical cell of a flow injection system. The dynamic range of the sensor was 20 to 160 ng/mL calpastatin. The electrode could be regenerated and re-used for more than 7 days with minimal reduction in sensitivity. For the analysis of real samples, the target analyte was extracted from the Longissimus dorsi muscle from beef carcasses directly after slaughtering. The extract was analyzed both with the developed immunosensor and microtiter plate ELISA, and a good correlation was obtained. However the immunosensor offers advantages of speed, simplicity, sensitivity and possibility for miniaturization over conventional assays for calpastatin quantification.

Characterization of GLUT4 and calpain expression in healthy human skeletal muscle during fasting and refeeding

Aims:  Calpain‐10 and calpain‐3 and the diabetes ankyrin repeat protein (DARP) have all been linked to insulin resistance and type 2 diabetes. We set out to measure the expression of these genes in human skeletal muscle and relate them to functional measurements of insulin action during fasting (which induces insulin resistance) and refeeding (which reverses it).

Principal component analysis of proteolytic profiles as markers of authenticity of PDO cheeses

The casein fraction of 13 Portuguese PDO cheeses were analysed using Urea-PAGE and reverse phase-high performance liquid chromatography (RP-HPLC) and then subjected to chemometric evaluation. The chemometric techniques of cluster analysis (CA) and principal component analysis (PCA) were used for the classification studies. Peptide mapping using Urea-PAGE followed by CA revealed two major clusters according to the similarity of the proteolytic profile of the cheeses. PCA results were in accordance with the grouping performed using CA. CA of RP-HPLC results of the matured cheeses revealed the presence of one major cluster comprising samples manufactured with only ovine milk or milk admixtures. When the results of CA technique were compared with the two PCA approaches performed, it was found that the grouping of the samples was similar. Both approaches, revealed the potential of proteolytic profiles (which is an essential aspect of cheese maturation) as markers of authenticity of PDO cheeses in terms of ripening time and milk admixtures not mentioned on the label.

Intron variability in an actin gene can be used to discriminate between chicken and turkey DNA

A novel one-step method for the differentiation of chicken and turkey DNA is described. The technique uses the polymerase chain reaction (PCR) and primers that exploit intron variability in α-cardiac actin to generate single products of a characteristic size for each species. No cross-reactivity with porcine, ovine or bovine DNA templates is apparent and analysis of chicken/turkey admixtures indicates that it is possible to detect 1% turkey in 99% chicken and vice versa. Because the test is based on a nuclear gene target, it forms a valuable complement to other methods based on mitochondrial DNA sequences.

Transient changes in growth and in calpain and calpastatin expression in ovine skeletal muscle after short-term dietary inclusion of cimaterol

Prolonged dietary inclusion of beta-adrenergic agonists can induce skeletal muscle hypertrophy in meat animals, by a mechanism probably related to the calcium-dependent proteolytic enzymes, or calpains, and in particular to their specific inhibitor calpastatin. Calpain and calpastatin activities are also believed to be important factors during post-mortem tenderisation of meat. beta-Agonist treatment is generally associated with increased calpastatin activity, which may lead to meat toughness. The aim of the present study was to examine the effect of a short period of cimaterol (feeding for 8 days, followed by reversion to a normal diet for a further 24 days) on muscle growth and on calpain isoform and calpastatin activities and specific mRNA abundance in the longissimus dorsi (LD) muscle. Significant changes were detected in LD wet weight and in calpastatin activity and mRNA after only 8 days treatment with cimaterol. After 24 further days on a control diet, both LD wet weight and calpastatin activity were not significantly different (P > 0.05) from untreated controls of the same age, although calpastatin mRNA stayed surprisingly high. In contrast to several earlier studies, changes in calpain I (or mu-calpain) and calpain II (or m-calpain) activity and calpain I mRNA were not significantly different (P > 0.05) from controls in any groups. These data suggest that calpastatin activity rather than the activity of either calpain isoform is closely linked to beta-agonist-induced muscle hypertrophy. Changes in calpastatin mRNA are not directly proportional to inhibitory activity, suggesting that variable mRNA species may be transcribed, spliced or stabilised, but not necessarily translated as part of the beta-agonist response.