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Poult Sci 2008. 87:2126-2132. doi:10.3382/ps.2007-00296
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PROCESSING, PRODUCTS, AND FOOD SAFETY

Role of Calpains in Postmortem Proteolysis in Chicken Muscle

H. L. Lee*,1, V. Santé-Lhoutellier{dagger},1, S. Vigouroux*, Y. Briand*,2 and M. Briand*,2

* Laboratoire de Génie Chimique et Biochimique, Unité Biochimie, Polytech’Clermont-Ferrand, Université Blaise Pascal, 63174 Aubière, France; and{dagger} INRA UR370, Qualité des Produits Animaux, 63122 Saint-Genès-Champanelle, France

2 Corresponding author: Yves.briand{at}univ-bpclermont.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Tenderness is governed by postmortem biochemical processes, particularly proteolysis. In mammals, the calpain system is generally accepted as the main system involved in postmortem proteolysis. In poultry, the 2 calpains (µ and µ/m – a form only found in bird tissue) have greater calcium sensitivity. In this study, we quantified by zymography the changes in postmortem calpain system activity. The µ/m-calpain activity remained steady, whereas the µ-calpain activity had disappeared by 6 h after postmortem, showing an activation by calcium. Changes in the electrophoretic pattern of sarcoplasmic and myofibrillar proteins are observed in the first postmortem hours concomitantly to the decrease in µ-calpain activity. The 30-kDa protein, considered as a good marker of postmortem aging in cattle, appeared from 6 h and then steadily increased. In chicken muscle, the rapid maximum tenderness reached could be explained by a greater activation of the calpain system.

Key Words: meat tenderness • chicken • postmortem proteolysis • calpain • muscle


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Consumers prize meat tenderness, which is the result of physicochemical and biochemical mechanisms acting mainly on myofibrillar structures postmortem. The kinetics of postmortem tenderization of muscle tissue have been well defined in cattle and sheep (Lepetit et al., 1986; Wheeler and Koohmaraie, 1994; Roncales et al., 1995). Muscles that are flexible and extensible at the death of the animal soon become rigid and enter rigor mortis, which corresponds to maximum toughness resulting from shortening of the myofibrils and in some cases lateral contraction (Offer, 1991; Wheeler and Koohmaraie, 1994; Koohmaraie et al., 1996). Over the same period, the pH decreases substantially. In a second, so-called postrigor phase, there is progressive tenderization of the meat resulting in organoleptic qualities acceptable to the consumer. Postmortem changes in tenderness are similar in different species, but the time-scale differs considerably. Rigor lasts more than 24 h in cattle and sheep, (Lepetit et al., 1986; Wheeler and Koohmaraie, 1994) but only 6 h in chicken breast muscle (Sams and Janky, 1991; Schreurs, 2000). Minimum toughness is reached after 8 d in cattle, whereas 18 h are enough in the chicken. Dransfield (1994) has shown that 80% of maximum tenderness can be reached only 0.3 d after slaughter in the chicken, whereas 4.2, 7.7, 9.5, and 10 d are needed in pig, sheep, rabbit, and cattle, respectively. It is generally accepted that the process of meat tenderization is essentially enzymatic and is dependent on the physicochemical conditions such as pH and ionic strength. Proteolysis of myofibrillar and myofibrillar-associated proteins tenderizes the meat by weakening intra- and intermyofibrillar bonds (Ouali, 1992; Koohmaraie, 1994, 1996; Taylor et al., 1995). The synergistic action of 3 proteolytic systems–cathepsins, the proteasome, and calcium-dependent proteases–has been proposed by several authors (Roncales et al., 1995; Robert et al., 1999; Sentandreu et al., 2002; Thomas et al., 2004; Ouali et al., 2006), but a whole range of studies show that the calcium-dependent system is essential for tenderization and reproduces in vitro all the phenomena observed postmortem (Koohmaraie, 1992; Huff Lonergan et al., 1996; Geesink and Koohmaraie, 1999; Geesink et al., 2000; Koohmaraie and Geesink, 2006). In mammals, where they have been studied in depth (Goll et al., 2003), there are 3 types of calpains in skeletal muscle: a tissue-specific calpain (p94), which is not involved postmortem (Geesink et al., 2005), and 2 ubiquitous calpains called calpains I and II, or µ- and m-calpain in reference to the calcium concentration required to activate them. These 2 enzymes are activated by partial autolysis in the presence of calcium. Prolonged autolysis then accounts for their inactivation (Li et al., 2004). In muscle postmortem, only µ-calpain is autolyzed as calcium is released and therefore seems to be involved in the tenderization mechanisms, unlike m-calpain, which remains intact for several days after slaughter because it requires high calcium concentrations (Koohmaraie et al., 1987; Geesink and Koohmaraie, 2000; Veiseth et al., 2001; Geesink et al., 2006). In birds, and in particular the chicken breast muscle, the role of postmortem proteolysis is poorly documented, and the few studies performed did not take into account the particularities of the calcium-dependent proteases in these species (McKee et al., 1997; Nurmahmudi and Sams, 1997; Veeramuthu and Sams, 1999; Obanor et al., 2005). In 1995, Sorimachi et al. found 4 mRNA coding for calpains in chicken breast muscle, corresponding, respectively, to the tissue-specific calpain p94, the ubiquitous µ- and m-calpains, all 3 of which are found in mammals, plus a fourth form not found in mammals. In chicken breast muscle, the form homologous to m-calpain is not translated, whereas the fourth form is translated into a calpain sensitive to calcium concentrations intermediate between those of mammalian m- and µ-calpains. For this reason, it was called µ/m-calpain (Sorimachi et al., 1995). We recently showed that the distribution of these 2 calpains in chicken varies from one tissue to another (Lee et al., 2007). In pectoralis superficialis muscle and iliotibialis muscle, the ratio of µ-calpain to µ/m-calpain is 1:10 [i.e., there is a marked predominance of µ/m-calpain (Lee et al., 2007)]. These chicken calpains are also more calcium-sensitive than mammalian calpains and may, therefore, play a special role in the postmortem process in chicken muscle, explaining its rapidity. We studied postmortem changes in these calpains and in parallel measured the activity of the proteasome, another protease likely to act in synergy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Bird and Tissue Samples

A total of 9 broiler chicken (8 wk old, 2.5 kg of live weight) were slaughtered at the abattoir of INRA Theix. Birds were electrically stunned (50 Hz, 120 mA, 4 s) and bled out. Three-gram samples of each pectoralis superficialis muscle were collected 5 min postmortem, 1 g was frozen in liquid nitrogen for biochemical assays, and 2 g was used for pH measurement. After evisceration, carcasses were chilled at 4°C, and 3-g samples of each pectoralis superficialis muscle were taken at different postmortem times (30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 24 h, 48 h, and 72 h), These samples were treated like the 5-min samples. Frozen samples were ground to powder and kept at –80°C until used. After 24 h, pectoralis superficialis muscles were removed from the carcass and placed on a tray wrapped with an air-permeable film and stored at 4°C.

pH Measurement

Two-gram samples of pectoralis superficialis muscle were taken at the different postmortem times (5 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 24 h) and homogenized in 18 mL of 0.005 M iodoacetate buffer using an Ultra-Turrax (Santé and Fernandez, 2000; IKA, Stanfer, Germany). The pH measurement was carried out 10 min after homogenization using a Xerolyt glass electrode (Mettler-Toledo, Viroflay, France) and a Geräte G800 pH meter (Schott, Mainz, Germany).

Preparation of Crude Extracts

For biochemical assays, muscle samples (200 mg) were homogenized in a Polytron homogenizer (Kinematica AG, Luzern, Switzerland; 19,000 rpm, 3 x 15 s in ice) in 2 mL of extraction buffer (50 mM Tris-HCl, pH 8.3, 20 mM EDTA, 10 mM ethylene glycol tetra-acetic acid (EGTA), and 0.1% mercaptoethanol). Homogenates were then centrifuged for 15 min at 10,000 x g, and the supernatant was centrifuged again under the same conditions. We used a muscle taken immediately after death (1 min) as a standard reference muscle, extracted in the same conditions as above, kept at –80°C. This reference sample was loaded on all the electrophoresis gels. Protein concentration was determined using Bio-Rad assay reagent (Bio-Rad, Hercules, CA) with bovine serum albumin as standard

Casein Zymography

Casein zymography was performed according to Raser et al. (1995) and Arthur and Mykles (2000), with some modifications (Lee et al., 2007), using the Mini-gel system (Bio-Rad). Resolving gels (10% acrylamide, 0.4% bisacrylamide in 375 mM Tris-HCl, pH 8.8) contained 0.2% casein (Hammerstein grade, ICN Biomedicals Inc., Costa Mesa, CA), and stacking gels (4% acryl-amide, 0.16% bisacrylamide in 330 mM Tris pH 6.8) contained no casein. Polymerization of both gels was catalyzed with 0.04% ammonium persulfate and 0.28% tetramethylethylenediamine.

A solution of 150 mM Tris-HCl pH 6.8, 20% glycerol (vol/vol), 0.75% mercaptoethanol (wt/vol), and 0.04% bromophenol blue was added to crude extracts (1/4 vol/vol). Standard reference muscle and samples (25 µg of protein) were subjected to electrophoresis at 100 V for 4 h at 4°C in 25 mM Tris-HCl, pH 8.3, 192 mM glycine, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT). After electrophoresis, the gels were rinsed (2 x 30 min, with gentle shaking) at 4°C in 20 mM Tris-HCl pH 7.5 containing calcium and then incubated for 18 h at 20°C in the same buffer containing 10 mM DTT. In the region of a band of activated calpain, the casein is digested into small fragments that diffuse out of the gel. The gels were stained for 2 h with Coomassie Brilliant Blue R and then placed in boiling water for 8 min, which gives a clear band in the presence of calpain. The bands were digitized by a Vitascan (Umax Data Systems Inc., Freemont, CA) using Photoshop software (Adobe Systems Inc., San Jose, CA). The resulting signals were quantified using QuantityOne software (Bio-Rad). Under our experimental conditions, the signal was proportional to the quantity of protein loaded on the gel.

Gel Electrophoresis

Subcellular Fractionation. Pectoralis superficialis muscle samples (1 g) were homogenized with a Polytron (low speed) in 10 mL of buffer A containing 50 mM KCl, 20 mM Tris pH 7.0, 2 mM EDTA, 4 mM MgCl2, 5 mM 2-mercaptomethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100 for 5 s. Buffer B containing 75 mM KCl, 10 mM KH2PO4, 2 mM MgCl2, 2 mM EGTA pH 7 was used for the last washes.

The homogenate was centrifuged at 10,000 x g for 10 min, and the supernatant (S1) was carefully decanted and saved. Ten milliliters of buffer B was added to the first pellet, and the homogenization was repeated. The centrifugation was repeated to obtain S1 to S3 and P3 fractions. The protein concentration of each fraction was determined using the Bradford procedure. Sarcoplasmic fractions were stored at –20°C before electrophoresis. Myofibrillar fractions were stored in buffer B containing 50% (vol/vol) glycerol before electrophoresis.

Electrophoresis Conditions. Polyacrylamide slab gels were run using a SE 250 Mighty Small unit (Hoefer Inc., Holliston, MA), using 11% resolving gels for both myofibrillar and sarcoplasmic proteins, which were prepared according to Fritz and Greaser (1991) and loaded at 10 µg of protein/lane. The reservoir buffer was that described by Laemmli (1970), and gels were run at 4°C using a constant current of 15 mA per gel. Gels were stained in a solution of 0.05% Coomassie Blue R250, 30% ethanol, and 5% acetic acid for 2 h and destained in a solution of 30% ethanol and 5% acetic acid. Gels were scanned and then analyzed with Sigma Gel software (Sigma-Aldrich Co., St. Louis, MO).

Proteasome Activity

Extracts were prepared according to Farout et al. (2003). Muscle (500 mg) was suspended (1:10 wt/vol) in 50 mM Tris-HCl buffer pH 8.0 containing 10% glycerol, 1 mM EDTA, 1 mM EGTA, 50 nM E64, 2.5 mM pepstatin A, and homogenized with a Polytron device. Crude extracts were prepared by centrifuging the homogenates at 100,000 x g for 1 h and were studied directly.

Chymotrypsin-like activity was measured using the fluorogenic substrate suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Sigma, St. Louis, MO). Reactions were performed in a final volume of 200 µL, containing 50 mM Tris-HCl (pH 8.0), 1 mM DTT, the sample, and 40 µM suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. After incubation for 30 min at 37°C, the reaction was stopped by the addition of 800 µL of 100 mM monochloroacetate-30 mM sodium acetate. The fluorescence was monitored in a F2000 fluorimeter (Hitachi Ltd., Tokyo, Japan), using 370 nm of excitation and 430 nm of emission.

Statistical Analysis

All data are expressed as mean ± SE and are representative of 5 to 9 experiments. Analysis of variance was carried out using the GLM procedure of SAS (SAS Institute, 1989). The model included effect of postmortem time, and means were compared using Duncan’s multiple range test. A value of P < 0.05 was taken as statistically significant.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Figure 1Go shows postmortem acidification of pectoralis superficialis muscle, which reflects the development of rigor mortis, and pH stabilization at a value called ultimate pH, at which the muscle enters rigor. In our conditions, the pectoralis superficialis muscle entered rigor 5 to 6 h postmortem, and thereafter, the pH did not change significantly. In sheep and cattle, the ultimate pH is reached on average 24 h after slaughter, at the same time as rigor mortis (Wheeler and Koohmaraie, 1994; Lepetit et al., 1986; Roncales et al., 1995; Lamare et al., 2002). Most studies in the chicken breast muscle have been done at early times (less than 3 h after slaughter) or later (24 h), and the postmortem time when the ultimate pH is reached is poorly defined (Veeramuthu and Sams, 1999; El Rammouz et al., 2004). Smith et al. (1969) suggested that pH could be a valuable tool to predict the development of rigor mortis, but it was only recently that Thielke et al. (2005), in 2 independent tests, showed that the ultimate pH was reached after 11 and 7 h, depending on the test, and that in parallel muscle, toughness peaked at 9 and 6 h, respectively. These authors did not, however, explain the observed differences between the 2 tests. Schreurs (2000) reported that chicken breast muscle toughness peaks 6 h after slaughter, which corresponds in our conditions to the time at which the ultimate pH is reached. These results show that the ultimate pH is reached simultaneously with rigor mortis, the onset of rigor mortis being earlier in chicken than in mammals. This could be explained by faster muscle metabolism and glycogen utilization in chicken breast muscle. Because minimum toughness is also reached faster in chicken, we investigated whether this specificity is linked to proteolysis.


Figure 1
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  		Figure 1. pH decline of chicken pectoralis muscle during meat aging. Vertical bars show SEM (n = 9).

 
Figure 2Go shows how the activities of the different calpain isoforms in chicken pectoralis superficialis muscle changed after slaughter. Casein zymography specifically measures the activities of the calcium-dependent proteases, without prior purification steps that could alter their activity (Lee et al., 2007). It can also be used to analyze simultaneously a large number of samples and is particularly suited to comparative studies. Gels were activated in a 100-µM calcium solution in which a signal proportional to each calpain isoform is obtained (Lee et al., 2007) Samples collected at different postmortem times from the same muscle were studied in the same gel and compared with a reference identical for all the broilers studied and taken as 100% (see materials and methods section).


Figure 2
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  		Figure 2. Casein zymography of calpains in chicken pectoralis muscle at various times postmortem. Std = Standard reference muscle; µ = µ-calpain; µ/m = µ/m-calpain.

 
In the reference muscle, there were 2 forms of calpain: µ-calpain and the µ/m-calpain specific to birds. The ratio of µ-calpain to µ/m-calpain was close to 1:10 (Lee et al., 2007) and was unchanged 5 min after slaughter compared with the standard reference muscle and then decreased greatly, because µ/m-calpain is very stable and had diminished little by 24 h postmortem (Figure 3AGo), whereas µ-calpain had strongly decreased by 6 h postmortem (Figure 3BGo). After 12 h, µ-calpain activity was hardly detectable. This could explain why avian muscle tenderizes rapidly. The 2 forms of calpain in birds are more calcium-sensitive than their mammalian counterparts, and it may be that µ-calpain is mobilized very soon after slaughter, because, for example, its calcium requirement is very low and below that of cattle µ-calpain. Jeacoke (1993) in mouse single muscle fiber has shown that the cellular free calcium concentration of 0.2 µM increases to a plateau of 100 µM 10 min postmortem, which is insufficient to activate mammalian m-calpain but enough to activate the 2 calpains in birds. Recently, Ji and Takahashi (2006) reported that the free calcium concentration in chicken muscle was 70 µM 20 min postmortem, 120 µM after 6 h, and 220 µM at 18 h, whereas more than 3 d were needed to reach a concentration above 200 µM in pigs and cattle.


Figure 3
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  		Figure 3. Caseinolytic activity of (A) µ/m-calpain and (B) µ-calpain in pectoralis muscle at different times postmortem. Activities are expressed as percentages of the reference muscle activity, which is taken as 100%. Vertical bars show SEM (n = 5 to 9). Different letters indicate significant differences.

 
Activation of µ-calpain could therefore occur very soon after slaughter, thus rapidly inducing proteolysis, which would initiate alteration of myofibrillar structure. Dransfield (1994) showed that 80% of maximum tenderness can be reached after 0.3 d of storage at 1°C. Our results show that µ-calpain had almost disappeared by 12 h postmortem, which roughly corresponds to the time determined by Dransfield for advanced tenderization.

A third band with calcium-sensitive proteolytic activity appeared during muscle aging (Figure 2Go). We have shown that this band is present in vivo in certain tissues, such as brain and liver, but not in muscle (Lee et al., 2007). This was confirmed by the standard reference muscle shown in Figure 2Go. After death, this activity was significant and reproducible after 6 h, when it accounted for 7% of the total calpain activity (µ-calpain and µ/m-calpain then represented, respectively, 4 and 89% of this total activity). This activity increased steadily to 32% of the total activity by 72 h, whereas µ-calpain became undetectable. It is possible that this third form has an increased specific activity, because the activity of µ/m-calpain, which is probably the source of this third form, was only decreased by 10%. The third form of calpain, which we suppose to be phosphorylated (Lee et al., 2007), appeared 24 h postmortem, whereas µ/m-calpain started to diminish slightly. This third form reached levels above those of µ-calpain, and its possible postmortem action should not be overlooked.

We used crude muscle extracts to test the autolysis of calpains, calcium sensitivity, and the presence of the third form, whose electrophoretic migration is fastest. The extracts were preincubated with 10 and 100 µM calcium for increasing times. After addition of EGTA + EDTA to stop calpain activity in extract, caseinolytic activities were measured by zymography. Figure 4Go shows that µ-calpain disappeared after 15 min of preincubation, whatever the calcium concentration, whereas about 30% of the initial µ/m-calpain activity remained (i.e., 70% of the µ/m-calpain had been autolyzed). No change was seen in the third form, regardless of the calcium concentration. These results clearly show that for 10 µM calcium, the concentration found in muscle postmortem, the 2 chicken calpains can be activated and autolyzed and so may play an important role in postmortem proteolysis. These calcium concentrations can easily be reached postmortem, and µ-calpain decreases from 24 h onwards (Figure 3Go).


Figure 4
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  		Figure 4. Autolysis of calpain in pectoralis muscle extracts. (A) Casein zymography of calpains after incubation of crude extracts in the presence of calcium for different times. Caseinolytic activity of (B) µ/m-calpain and (C) µ-calpain expressed as a percentage of the activity at time 0, which is taken as 100%. Vertical bars show SEM (n = 5 to 9).

 
The chymotrypsin-like activity of the proteasome postmortem was measured in the same samples as those shown in Figure 2Go and 3Go. This activity was chosen, because it is representative of the quantity of proteasome (Farout et al., 2003). This second proteolytic system may also participate actively in proteolysis. Our results show that although its activity decreased faster than in cattle (Lamare et al., 2002), 69% of the initial activity remained after 6 h of storage and 36% after 72 h (Figure 5Go), which is probably enough to complement the calpains whose activity initially destabilizes and disrupts the myofibrillar structures, slightly denaturing the constituent proteins and thus allowing the proteasome to act. This is necessary, because the proteasome cannot hydrolyze native proteins but can use partially denatured proteins as substrate. Calpains have a limited hydrolytic action on proteins, and the absence of hydrolytic products, apart from the 30-kDa peptide, suggests that they have probably been hydrolyzed by the proteasome. Thomas et al. (2004) have shown that the proteasome is likely involved in the tenderization of ostrich meat.


Figure 5
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  		Figure 5. Chymotrypsin-like activity of proteasome in chicken pectoralis muscle. Activities are expressed in nanomoles of substrate hydrolyzed per milligram of protein per 30 min. Different letters indicate significant differences.

 
Figure 6Go shows the electrophoretic patterns of the myofibrillar and sarcoplasmic proteins of chicken pectoralis superficialis muscle obtained at different times postmortem. For the myofibrillar proteins (Figure 6AGo), 3 important modifications can be observed. One protein of molecular weight near 110 kDa disappeared between 3 and 6 h postmortem, whereas 2 proteins of approximately 105 and 30 kDa appeared. The 110-kDa protein could be {alpha}-actinin, which is known to be a substrate of calpains. The 30-kDa protein appeared between 6 and 12 h and then steadily increased. This protein has been observed postmortem in mammals and results from the partial hydrolysis of troponin T, probably by calpain (MacBride and Parrish, 1977; Olson et al., 1977; Huff Lonergan et al., 1996; Negishi et al., 1996). This 30-kDa protein is considered to be a good marker of postmortem aging in cattle (MacBride and Parrish, 1977; Penny and Dransfield, 1979) and has also been found in chicken breast and thigh muscles (Hay et al., 1973). The proteolytic pattern did not change after 24 h. Postmortem proteolysis also affects sarcoplasmic proteins, because the band around 80 kDa intensified with time and the 75-kDa band decreased after 6 h postmortem (Figure 6BGo).


Figure 6
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  		Figure 6. Sodium dodecyl sulfate gel electrophoresis of (A) myofi-brillar and (B) sarcoplasmic proteins of chicken pectoralis muscle at different times postmortem.

 
In conclusion, the situation in the chicken pectoralis muscle seems to differ substantially from that in mammals, in which µ-calpain disappears from 24 h after slaughter, the aging process continues for several days, and m-calpain is still inactive because the calcium concentration is too low to activate it. The required calcium concentration is only reached 10 d after slaughter, and Geesink et al. (2000) have discounted the involvement of m-calpain in the tenderization process in mammals, in which the proteasome may take over when µ-calpain disappears, as suggested by Lamare et al. (2002).

In the chicken pectoralis muscle, the rapid intervention of calcium-dependent proteases and the resulting early appearance of the 30-kDa peptide explain the rapid aging seen in this species. Other proteases, which are numerous in skeletal muscle, may also be involved, and Blanchard and Mantle (1996) have shown that these proteases are all more active in the breast and thigh muscles of chicken than of other species such as pig, sheep, and rabbit, in which postmortem aging of meat is slower.


   FOOTNOTES
 
1 The first two authors have equally contributed to this work. Back

Received for publication July 19, 2007. Accepted for publication June 13, 2008.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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