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The relationship between meat tenderization, myofibril fragmentation and autolysis of calpain 3 during post-mortem aging
Meat Science 66 (2004) 387–397 www.elsevier.com/locate/meatsci
The relationship between meat tenderization, myofibril fragmentation and autolysis of calpain 3 during post-mortem aging Mohammad A. Ilian*, Alaa El-Din Bekhit, Roy Bickerstaffe Molecular Biotechnology Group, Animal and Food Sciences Division, PO Box 84, Lincoln University, Canterbury, New Zealand Received 21 November 2002; received in revised form 4 March 2003; accepted 22 April 2003
Abstract The objective was to study the potential role of calpain 3 in postmortem myofibril breakdown and meat tenderization. We determined the temporal changes in calpain 3 protein in the ovine m. longissimus thoracis et lumborum (LTL, n=4) during postmortem storage. Concurrently, we also determined the kinetics of tenderization level, changes in MFI, degradation of nebulin and desmin, and autolysis of calpain 1. The autolysis of calpains 1 and 3 were strongly correlated with the kinetics of tenderization and changes in MFI. The best correlation was between the appearance of the autolyzed calpains 1 and 3 and nebulin degradation. Taken together, the results indicated that calpains 1 and/ or 3 might be playing a key role in post-mortem tenderization of LTL via the proteolysis of specific muscle structural proteins such as nebulin. This is the first report that relates calpain 3 to myofibrillar protein degradation in post-mortem skeletal muscle. # 2003 Elsevier Ltd. All rights reserved. Keywords: Skeletal muscle; Tenderness; Proteolysis; Calpain 3
1. Introduction Calpains (EC 3.4.22.17) constitute a large family of intracellular Ca2+-dependent cysteine neutral proteinases which are believed to be involved in many Ca2+-regulated physiological as well as pathological conditions (Sorimachi, Ishiura, & Suzuki, 1997; Huang & Wang, 2001). The calpains are classified on the basis of tissue distribution into ubiquitous and tissue-specific. At the last count, the calpain proteolytic system in mammalian skeletal muscle is composed of eight ubiquitous (calpains 1, 2, 5, 7, 10, 12, 14 and 15) and one tissue-specific (calpain 3) isoenzymes. Dayton, Goll, Zeece, Robson, and Reville (1976) purified the first calpain (calpain 2) from porcine skeletal muscle. Since that time, a great deal of experimental evidence has accumulated, indicating that the calpain proteolytic system has a primary role in myofibrillar protein degradation (Barnoy, SupinoRosin & Kosower, 2000; Goll, Thompson, Taylor, &
* Corresponding author. Tel.: +64-3-3253803; fax: +64-3-3253851. E-mail address: [email protected] (M.A. Ilian). 0309-1740/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0309-1740(03)00125-6
Christiansen, 1992; Huang & Forsberg, 1998; Poussard et al., 1996). In post-mortem skeletal muscles of meat animals, the proteolytic activity of the calpain system on the myofibrillar proteins has the primary influence on meat tenderness (Bickerstaffe, 1996; Koohmaraie, 1992; Ouali, 1992). Therefore, tangible benefits could be achieved by understanding the mechanism of meat tenderization. Complete understanding of the biochemical basis of meat tenderization requires the identification of the calpains associated with this process and their protein substrates. Calpains 5, 7, 10, 12, 14 and 15 were recently discovered (Dear & Boehm, 1999; Dear, Meier, Hunn, & Boehm, 2000; Franz, Vingron, Boehm, & Dear, 1999; Horikawa et al., 2000; Kamei, Webb, Young, & Campbell, 1998) and their biochemical role in skeletal muscle and meat is unknown. A recent conference review paper (Geesink, Ilian, Morton, & Bickerstaffe, 2000) on the role of calpains 1, 2 and 3 in meat tenderization concluded the following: ‘‘1) Post-mortem tenderization is primarily caused by m-calpain (calpain 1)-mediated degradation of key muscle structural proteins. 2) Unless meat is treated with mM levels of calcium, m-calpain
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(calpain 2) appears not to be involved in post-mortem tenderization. 3) The role of calpain 3 in tenderization remains to be determined’’. A very recent comprehensive review on current theories of tenderization of red meat concluded that the major candidate to explain tenderization post-rigor is the calpain system and pointed out that among the issues that remain to be resolved is the contribution of calpain 3 to tenderization (Hopkins & Thompson, 2002). The interest in calpain 3 with regard to postmortem tenderization of meat stems from the observations that calpain 3 is a muscle-specific calpain (Sorimachi et al., 1989) and that it binds to titin at the N2 line, a site where proteolysis in the early post-mortem period has been linked to tenderization (Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). The reports on the role of calpain 3 in meat tenderization are very limited due to constraint of investigative tools. With the development of analytical methods for the determination of calpain 3 expression at the mRNA and protein levels some information on the role of calpain 3 in meat tenderization has been published. The first publication on the role of calpain 3 in meat tenderization was presented by Parr et al. (1999) who did not find a correlation between variations in porcine m. longissimus thoracis et lumborum (LTL) tenderness and variations in the level of calpain 3. By comparison, our previous studies on the relationship between calpain 3 and meat tenderization revealed, for the first time, a strong correlation between the variations in ovine meat tenderness and the expressions of calpain 3 at the protein (Ilian et al., 2001a) and mRNA (Ilian et al., 2001b) levels. The objective of this study was to gain insight into the biochemical role of calpain 3 in meat tenderization in comparison with calpain 1. We determined the temporal changes in the protein status of calpain 3 in the ovine LTL during postmortem storage. Concurrently, we also determined the temporal changes of; (1) tenderization level, (2) myofibrillar fragmentation index (MFI), (3) proteolysis of nebulin and desmin, and (4) autolysis of calpain 1. The relationship between calpain 3, meat tenderization and myofibrillar protein degradation during post-mortem storage is discussed on the basis of the results obtained.
2. Materials and methods 2.1. Animals and sample collection Four female Coopworth lambs, age 4.5 months were slaughtered by captive-bolt stunning and exanguinated and dressed at the Lincoln University large animal anatomy laboratory. A sample ( ffi 7g) from the right side LTL was extracted from the L7 vertebra area about 15 min after killing and snap frozen in liquid nitrogen for Western analysis of nebulin, desmin and calpains 1
and 3. The carcasses were kept for 4 h at 15 C and then moved to a 2 C chiller for 7 days. At 1, 2, 3, 4, 5, 6 and 7 days post-mortem, a sample ( ffi 7g) and a steak (about 5 cm5 cm) from the right side LTL were removed from the L6-5, L4-3, L2-1, T13-12, T11-10, T9-7, T6-4 regions, respectively for laboratory analyses. The samples were immediately frozen in liquid nitrogen. The steaks were placed in plastic bags and stored in a freezer at 20 C. 2.2. Tenderness assays Frozen steaks were thawed overnight at 2 C and LTL tenderness was determined subjectively by measuring the force required to shear through a cooked sample as was described earlier (Ilian et al., 2001b). 2.3. Myofibril fragmentation index The myofibril fragmentation index (MFI) was determined on frozen samples taken at 1, 2, 3, 4, 5, 6 and 7 days of post-mortem according to the method described by Culler, Parrish, Smith, and Cross (1978) with some modification. This included using a Polytron1 (Kinemacia, Littau, Switzerland) at 10,000 rpm for 30 s to homogenize the tissue. 2.4. Western blot assays To study the kinetics of the proteolysis of nebulin and desmin and the autolysis of calpains 1 and 3 during post-mortem storage, we separated the LTL proteins from the samples taken after 0, 1, 2, 3, 4, 5, 6 and 7 days of post-mortem storage into sarcoplasmic and myofibrillar fractions. This was performed according to the method described by Boehm, Kendall, Thompson, and Goll (1998) with some modification (Ilian et al., 2001a). Protein concentration of the various fractions was determined according to Karlsson, Ostwald, Kabjorn, and Anderson (1994). Nebulin SDS–PAGE was performed according to Huff-Lonergan, Parrish, and Robson (1995) to allow for the separation of high molecular weight proteins. Other proteins were separated on SDS– PAGE (stacking gel, 3.5% polyacrylamide; separating gel, 8% polyacrylamide) using mini-gels according to Laemmli (1970). Western blot assays of myofibrillar and sarcoplasmic fractions were conducted by loading 27.5 and 9.1 mg protein respectively together with pre-stained broad range SDS–PAGE standards (BIO-RAD, CA). After electrophoresis, gels were electroblotted on polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK) at 4 C overnight and 30 V using a trans-blot unit (BIO-RAD, CA) according to Towbin, Staedhin, and Gordon (1979). The transfer buffer (pH 8.3) was 25 mM Tris, 192 mM glycine, 15% methanol, and 0.5% SDS. Gels
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after transfer were stained with Coomassie Blue to examine the efficiency of transfer. Primary antibodies used in the Western blot assays were as following. (1) Monoclonal anti-nebulin NB2 clone at 1:250 (Sigma Chemical, St. Louis, Mo). (2) Monoclonal anti-desmin Clone D3 at 1:100 (developed by D. A. Fishman, Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242 under the contract N01-HD7-3263 from the NICHD). (3) Monoclonal anti-calpain 1 clone MA3-941 at 1:1000 (Affinity Bioreagents, Golden, CO). (4) Polyclonal anti-calpain 3 Calp3IS2:b/o at 1:500 and a description of the preparation and purification of the Calp3IS2:b/o was given previously (Ilian et al., 2001a). The specificity of all antibodies used in this study for the analyses of their respective antigen was confirmed by producing the respective signals for their antigens on Western blot. The quantitative aspect of the assays of antibodies used in this study for the analyses of their respective antigen were determined by constructing a calibration curve, which showed a linear response of image density to gradation in LTL protein level (results not shown). Primary and secondary antibodies were diluted in blocking buffer (100 mM Tris–Cl, pH 7.5, 0.9% NaCl, 0.1% Tween 20, 5% nonfat milk powder) according to Towbin et al. (1979). After washing, detection of bound antibodies was performed with a chemiluminescence kit (Pierce, IL) and the intensity of signal was determined using a UVP gel documentation system (UVP Inc. Upland, CA). 2.5. Analyses of data Temporal improvements in LTL tenderness, designated here as tenderization level, for each animal (n=4) with time were calculated as a percentage of improvement in tenderness at day 7 post-mortem using shear force value at day 1 as a baseline. The equation for calculating tenderization level was ðkgf 1 kgfn Þ ðkgf 1 kgf 7 Þ 100 where kgfn is shear force value at day n. To determine the effect of time on LTL tenderization level and MFI, data were fitted to an exponential rise to maximum (single variable, three constants) model using the equation Y ¼ Y0 þ a 1 ebX with LTL tenderization level or MFI as the response and time post-mortem as the predictor. Also, one way ANOVA was performed using tenderization level and MFI values as dependent variables and time as the factor level to detect significant differences among the means. To determine the relationship between tenderization level and MFI we performed regression analysis by fitting a linear regression model to the data using the equation Y ¼ 0 þ 1 X with MFI as the response and tenderization level as the predictor. To study the effect of time on the proteolysis of
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nebulin and desmin and the autolysis of calpains 1 and 3 during post-mortem storage the data for each protein from each animal were normalized as percentages of the value at slaughter. Then the normalized values of 1–7 days post-mortem were fitted to an exponential decay (single variable, three constants) model using the equation Y ¼ Y0 þ a ebX with protein level as the response and time post-mortem as the predictor. The exponential regressions provided a means for showing the relationship between myofibril fragmentation and autolysis of calpains 1 and 3. To evaluate the relationship between tenderization level, MFI, proteolysis of nebulin and desmin and autolysis of calpains 1 and 3, the Pearson product moment correlation coefficient between each pair of the above variables was calculated. The software used for performing the above statistics and curve fitting was Minitab version 11 computer package (State College, PA).
3. Results and discussion 3.1. Specificity of anti-calpain 3 antibody Quantitative determination of calpain 3 expression is very important in the study of its function in skeletal muscle and meat. Unlike the ubiquitous calpains 1 and 2, analysis of the activity of calpain 3 is not achievable at the present time because of its propensity to undergo very rapid autolysis during extraction (Kinbara et al., 1998). Having said that, this does not mean that alternative approaches are not available to conduct meaningful research on the role of calpain 3 in meat tenderization. The expression of the calpain proteolytic system at the mRNA (Ilian et al., 2001b; van den Hemel-Grooten, Te Pas, van den Bosh, Garssen, Schreurs, & Versegen, 1997) and protein levels (Ilian et al., 2001a; Parr et al., 1999) have often been determined to understand their role in muscle functioning. In this study, established immunochemical techniques were employed to study the biochemical pathway of meat tenderization during post-mortem storage and the role of calpain 3 in this process. The validity of analysis of calpain 3 in this study absolutely depends on the specificity of the antibody. Details for the development and validation of our anticalpain 3 antibody Calp3IS2:b/o for western assay of bovine and ovine calpain 3 were reported earlier (Ilian et al., 2001a). In brief; 1. We have sequenced the bovine and the ovine calpain 3 cDNA and used the IS2 region, which is unique to calpain 3, for designing the antigenic polypeptide of Calp3IS2:b/o. 2. We have conducted a BLAST search to examine the homology of the antigenic polypeptide of
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Calp3IS2:b/o to other proteins. The results indicated that the antigenic peptide is unique to calpain 3 and not present in other known proteins. 3. Based on published results, calpain 3 is a skeletal muscle specific enzyme with 94 kDa molecular weight (Sorimachi et al., 1989). We utilized this information to examine the specificity of Calp3IS2:b/o. We tested Calp3IS2:b/o with proteins from skeletal muscle, liver, heart and lung. Calp3IS2:b/o only reacted with proteins from skeletal muscle and produced a signal at 94 kDa (Ilian et al., 2001a). 4. Furthermore, we tested the specificity of Calp3IS2:b/o using pure p94:C129S. Protein p94:C129S is a calpain 3 mutant in which the active site (Cys-129) had been changed to serine to eliminate the protease activity (Kinbara et al., 1998). Calp3IS2:b/o reacted with p94:C129S very strongly. Essentially the same signal was obtained with the skeletal muscle (Ilian et al., 2001a). 5. During the preparation of the manuscript, Koohmaraie, Kent, Shackelford, Veiseth, and Wheeler (2002) described unpublished work from Goll laboratory indicating their calpain 3 antibodies reacted with 94 kDa non-calpain proteins (determined by micro-sequencing). Therefore the Calp3IS2:b/o antibody was tested against two prominent muscle proteins of this approximate size, phosphorylase and phosphofructokinase (PFK). A blot using purified phosphorylase a and b, PFK, (Sigma Australia) plus skeletal muscle and the p94:C129S protein as positive controls is shown in Fig. 1. The results indicated that Calp3IS2:b/o labeled p94:C129S and skeletal muscle but did not label phosphorylases a and b or PFK even at the high concentration used (200 ng). The explanation for the faint trail in the p94:C129S lane of Fig. 1 is that during the purification of this mutant inactive calpain 3,
Fig. 1. SDS–PAGE of skeletal muscle phosphorylase a and b, skeletal muscle phosphofructokinase, p94:C129S pure proteins together with whole ovine longissimus proteins and Western analysis of same using Calp3IS2:b/o.
the proteolytic enzymes of the COS cells caused limited degradation of the p94:C129S (Sorimachi, personal communication). No implications of using p94:C129S for the validation of the specificity of Calp3IS2:b/o for the detection of native calpain 3 are anticipated. This is because Calp3IS2:b/o was raised against an epitope in the IS2 region, which is distant from the active site region.
3.2. Tenderization and MFI profiles The kinetics of tenderization of LTL (expressed as percentage of the tenderization at 7 days) over a period of 7 days post-mortem storage under refrigerated conditions are presented in Fig. 2a. Presentation of shear
Fig. 2. Effect of postmortem storage period on tenderness and myofibrillar fragmentation index in ovine m. longissimus thoracis et lumborum (LTL). Kinetics of tenderization and myofibrillar fragmentation index of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions are presented in panels a and b, respectively. Regression analysis between the results of tenderization level and MFI during 1–7 days post-mortem is shown in panel c.
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force values as tenderization level was done for the following reasons. (1) To normalize the results across the experimental animals used in the study. (2) To provide a clearer relationship between changes in LTL tenderness and time of post-mortem storage. (3) To provide a direct, not inverse, mathematical relationship between tenderness and myofibrillar fragmentation index. The results indicate that significant improvements in LTL tenderness occurred gradually during the first three days of post-mortem storage. On average, 44 and 72% improvements over the 1 days tenderness values were observed at days 2 and 3 post-mortem, respectively. This is in agreement with previous results reported by our laboratory (Ilian et al., 2001b) and others (Wheeler & Koohmaraie, 1994). To gain insight into whether tenderization of LTL is primarily due to myofibrillar protein degradation, we determined the kinetics of MFI over a period of 7 days post-mortem storage under refrigerated conditions as shown in Fig. 2b. MFI is a useful indicator of the extent of myofibrillar protein degradation (Olson, Parrish, & Stromer, 1976). Significant increases in MFI occurred gradually during days 2 and 3 of post-mortem storage compared with day 1. To determine the relationship between tenderization level and MFI we fitted a linear regression model to the data using the equation Y ¼ 0 þ 1 X with MFI as the response and tenderization level as the predictor (Fig. 2c). The results revealed a strong correlation (r=0.787, P40.01) between the kinetics of tenderization and MFI indicating that myofibrillar protein degradation during post-mortem storage of LTL was closely associated with tenderization in this study.
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Fig. 3. Kinetics of nebulin proteolysis in the myofibrillar fraction of ovine LTL (n=4) during 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of nebulin proteolysis.
3.3. Proteolysis of nebulin and desmin As a confirmation for the MFI results we examined myofibrillar protein degradation in LTL during postmortem storage at the molecular level. Western blot assays were used to measure the kinetics of proteolysis of nebulin and desmin in LTL over a period of 7 days post-mortem storage under refrigerated conditions (Figs. 3 and 4). Nebulin and desmin were investigated for the following reasons. Firstly, they play important roles in organizing and maintaining the integrity and strength of the contractile myofibrils and the overall cytoskeleton structure of the skeletal muscle cell (Robson et al., 1997). Nebulin and desmin are also known to undergo post-mortem proteolysis at a fast and slow rate respectively (Robson et al., 1997). Finally, they are excellent substrates for the calpain proteolytic system (Huff-Lonergan, Mitsuhashi, Beekman, Parrish, Olson, & Robson, 1996). Kinetics of nebulin proteolysis in LTL (as percentage of value at slaughter) over a period of 1–7 days postmortem storage at 2 C is presented in Fig. 3. Essentially no fragmentation of nebulin occurred during the
Fig. 4. Kinetics of desmin proteolysis in the myofibrillar fraction of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of desmin proteolysis.
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first 24 h post-mortem. However, complete fragmentation of nebulin occurred by 3 days post-mortem. After 2 and 3 days post-mortem, about 70 and 97% of intact nebulin was degraded, respectively. The kinetics of desmin proteolysis in LTL over a period of 1–7 days post-mortem storage at 2 C is presented in Fig. 4. As with nebulin, very limited degradation of desmin occurred during the first 24 h post-mortem. The rate of degradation of desmin, however, is much slower than that of nebulin. After 2 days post-mortem, some desmin was degraded but the results were not significantly different from the level at slaughter. Significant amounts of desmin were degraded after 3 and 5 days post-mortem storage with losses of 33 and 71% of intact desmin, respectively. Therefore, the above results confirm the results of MFI and indicate that the biochemical basis of LTL tenderization during post-mortem aging in this study is the breakdown of certain myofibrillar proteins including nebulin and desmin. However, the rate of degradation, in agreement with others (Robson et al., 1997), depends upon storage time and protein species. 3.4. Autolysis of calpains It is believed that the calpain proteolytic system has more than one mechanism of activation depending on the cellular condition and that autolysis is perhaps one of the mechanisms of calpain activation (Molinari, Anagli, & Carafoli, 1994; Saido, Suzuki, Yamazaki, Tanoue, & Suzuki, 1993; Suzuki, Tsuji, Kubota, Kimura, & Imahori, 1981). Several aspects of activity regulation of calpains in relation to autolysis have been investigated. These include; the effect of autolysis on Ca2+ sensitivity (Baki, Tompa, Alexa, Molnar, & Friedrich, 1996; Goll, Thompson, Taylor, & Zalewska, 1992), substrate specificity (Schoenwaelder et al., 1997), subunits interaction and proteolytic activity (Suzuki & Sorimachi, 1998). It is important to indicate that autolysis of calpains was observed in pathological conditions associated with aberrant rise in the concentration of intracellular calcium (Vanderklish & Bahr, 2000). The most clearly documented biological relevance of calpain autolysis in the literature does not involve a function in living cells, but rather its role in postmortem tenderization of meat. Koohmaraie, Seideman, Schollmeyer, Dutson, and Crouse, (1987) proposed that calpain 1, and not calpain 2, is responsible for meat tenderization because its activity gradually declined during post-mortem aging of meat. Boehm et al. (1998) conducted a rigorous study on the changes in activity and protein structure of calpains 1 and 2 in bovine skeletal muscle during the first 7 days of post-mortem storage. They determined that extractable calpain 2 activity decreased slightly during the first 7 days post-mortem however, calpain 1 activity decreased to less than 4% of
at-death value. Western blot and sequencing analyses showed that the 80 kDa subunit of calpain 2 remained intact during the first 7 days postmortem however the 80 kDa subunit of calpain 1 was almost entirely converted to the 76 kDa autolyzed form. The authors concluded that calpain 2 is not proteolytically active during post-mortem storage of meat because it was not autolyzed. In a recent study, Veiseth, Shackelford, Wheeler, and Koohmaraie, (2001) utilized casein zymography to study the autolysis of calpains 1 and 2 in ovine meat during postmortem storage. Their results revealed that only calpain 1 showed autolysis under post-mortem conditions. A conservative interpretation of the above data is that autolysis indicates that a calpain has been activated, although subsequent modifications may cause inactivation. Thus, autolysis can be used as an assay of activation, but not activity (Tidball & Spencer, 2000). In this study, we determined the effect of post-mortem storage on the protein status of calpains 1 and 3. Calpain 1 was used for comparative purposes. An important aim here was to determine the association of calpain 3, like calpain 1, with LTL myofibrillar proteins. The underlying hypothesis was that in order for a protease to be involved in meat tenderization it must have access to the substrate and be associated with the myofibril. The myofibrillar-bound calpain 1 has been the subject of recent publications (Boehm et al., 1998; Delgado, Geesink, Marchello, Goll, & Koohmaraie, 2001). Western blot analyses of the large subunit of calpain 1 in the sarcoplasmic and myofibrillar fractions of LTL during a period of 7 days post-mortem storage are presented in Figs. 5 and 6, respectively. The results indicate that the catalytic subunit of calpain 1 is present in both the sarcoplasmic and myofibrillar fractions at slaughter primarily in the form of the 80 kDa form. Subsequently, the calpain 1 native catalytic subunit in both the sarcoplasmic and myofibrillar fractions was gradually autolyzed in the LTL during post-mortem storage. The autolysis kinetics of sarcoplasmic calpain 1 catalytic subunit, measured as the abundance of the 80 kDa subunit, indicate that the enzyme was autolyzed after slaughter and lost about 50 and 85% of its native form by 1 and 2 days post-mortem, respectively (Fig. 5). In contrast, the concentration of myofibrillar calpain 1 native catalytic subunit was not changed at 1 day postmortem compared with the initial value at slaughter (Fig. 6). Subsequently, myofibrillar calpain 1 native catalytic subunit autolysis was steep as 75% of the 80 kDa catalytic subunit was autolysed by 2 days postmortem. The autolysis pattern and localization of the native sarcoplasmic and myofibrillar calpain 1 catalytic subunit may indicate different functions. Logically, myofibrillar calpain 1 may be the one involved in myofibrillar protein degradation during post-mortem aging because of its association with the myofibrils.
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Fig. 5. Kinetics of autolysis of the catalytic subunit of calpain 1 in the sarcoplasmic fraction of ovine LTL (n=4) during a 7 days postmortem storage period under refrigerated conditions. Upper panel is a representative Western blot of calpain 1 catalytic subunit in the sarcoplasmic fraction.
Fig. 6. Kinetics of autolysis of the catalytic subunit of calpain 1 in the myofibrillar fraction of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of calpain 1 catalytic subunit in the myofibrillar fraction.
The propensity of calpain 3 to autolysis has been clearly documented (Kinbara et al., 1998). Early literature indicated that calpain 3 is destroyed by autolysis with a half-life of approximately 30 min in COS cells (Sorimachi et al., 1993). Recent studies, however, have shown that the above phenomenon is applicable to calpain 3 in skeletal muscle tissue but with much slower rates. For example, calpain 3 protein was found to be stable in fresh human muscle, with full-size protein being detected 8 h after the muscle had been removed (Anderson et al., 1998). Jones, Parr, Sensky, Scothern, Bardsley, and Buttery (1999) explained that calpain 3 autolyzes rapidly when expressed in COS cells, however, in vivo the enzyme may be stabilized by interaction with titin. Last but not least, intact calpain 3 protein in porcine longissimus muscle could be detected up to 24 h after slaughter in carcasses held in a chiller (Parr et al., 1999). In a previous study, we showed autolysis of calpain 3 in post-mortem skeletal muscle during aging (Ilian et al., 2001a). The autolytic process of calpain 3 in ovine skeletal muscle during post-mortem aging was characterized by a gradual increase in the concentration of a 52.5 kDa autolytic fragment in association with a corresponding gradual decrease of the level of intact 94 kDa protein. Unlike calpains 1 and 2, the kinetics of
autolyzis of calpain 3 and its physiological significance during post-mortem aging of meat has not yet been established. The observations that the three autolysis sites in calpain 3 are present in the catalytic domain II (Kinbara et al., 1998) and that mutations in the autolysis sites were reported in limb girdle muscular dystrophy type 2A patients (Beckmann et al., 1996) are indications that autolysis may be an important aspect of a functional calpain 3. Furthermore, Kinbara and associates (1998) indicated that the ffi 55 kDa fragment (in our study 52.5 kDa) is the C-terminus fragment of calpain 3 which comprises domain III and VI. Similar information on the nature of the ffi 55 kDa fragment of calpain 3 was given by Branca, Gugliucci, Bano, Brini, and Carafolli, (1999). It is important to indicate that the active site of calpain 3 is located in domain II (Sorimachi et al., 1989) and therefore, the 52.5 kDa fragment is expected to possess no proteolytic activity. In this study, the results of western blot analysis indicated that calpain 3 was present in the sarcoplasmic and myofibrillar fractions at slaughter and that calpain 3 in both fractions expressed autolysis in LTL during postmortem storage (Figs. 7 and 8). The kinetics of autolysis of sarcoplasmic calpain 3 indicate that the enzyme was terminally activated after slaughter and that about 40
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Fig. 7. Kinetics of autolysis of calpain 3 in the sarcoplasmic fraction of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of calpain 3 autolysis in the sarcoplasmic fraction.
and 85% of the native calpain 3 level was lost by 1 and 2 days post-mortem, respectively (Fig. 7). Similarly, the myofibrillar calpain 3 was autolyzed, but at a lower rate, after slaughter and about 25 and 75% of the 94 kDa protein was lost by 1 and 2 days post-mortem, respectively. Afterward, calpain 3 autolysis in the sarcoplasmic and myofibrillar fractions continued, but at a much lower rate, until 5 days post-mortem. 3.5. Calpains and post-mortem meat tenderization It is well established that post-mortem meat tenderization is a complex biochemical process involving the fracturing of key myofibrillar proteins which are responsible for maintaining the structural integrity of the myofibrils by the calpain proteolytic system (Hopkins & Thompson, 2002). However, the mode of action of the calpains in this process is not fully understood. The relative contribution of the various calpains in postmortem tenderization of meat is an open question (Boehm et al., 1998). Detailed understanding of the biochemical role of the calpain proteolytic system in meat tenderization requires the identification of the calpains associated with this process and their protein substrates. Previously, we have examined the role of
Fig. 8. Kinetics of autolysis of calpain 3 in the myofibrillar fraction of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of calpain 3 autolysis in the sarcoplasmic fraction.
calpain 3 in meat tenderization using three biological systems. The results, for the first time, have indicated that variations in the expression of calpain 3 at the mRNA and protein levels are strongly correlated to variations in the tenderness of post-mortem skeletal muscle (Ilian et al., 2001a, 2001b). Also the variations in the rate of titin proteolysis due to stretching is directly related to the rate of calpain 3 autolysis (Stevenson, Morton, Ilian, & Bickerstaffe, 2002). To gain insight into the mode of action of calpain 3 in post-mortem meat tenderization we performed correlation analyses for the temporal changes in tenderization level, nebulin and desmin proteolysis and autolysis of calpains 1 and 3 during 1–7 days of post-mortem storage (Table 1). The results indicate that kinetics of tenderization level is Table 1 Correlation matrix for temporal changes in tenderness level, nebulin and desmin proteolysis and calpains 1 and 3 autolysis during 7 days postmortem storage Tenderness Nebulin Desmin Calpain level proteolysis proteolysis 1(M)a autolysis Nebulin proteolysis Desmin proteolysis Calpain 1(M) autolysis Calpain 3(M) autolysis a
M, myofibrillar.
0.932 0.511 0.986 0.921
0.634 0.956 0.930
0.610 0.654
0.986
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highly correlated with the kinetics of nebulin proteolysis (r=0.932, P40.001) and the kinetics of myofibrillar calpains 1 (r=0.986, P40.001) and 3 (r=0.921, P40.001) autolysis. The correlation coefficient between the kinetics of tenderization level and desmin proteolysis was not significant (r=0.511, P40.05). However, this result does not discard a role for desmin proteolysis in the development of tenderness during post-mortem storage. These results suggest that nebulin proteolysis may be a key component in tenderization of meat. Furthermore, the kinetics of nebulin proteolysis was strongly correlated with the autolysis of the catalytic subunit of the myofibrillar calpain 1 (r=0.956, P40.001) and myofibrillar calpain 3 (r=0.930, P40.001). These results provide indirect evidence that calpains 1 and/or 3 may be responsible for the fragmentation of nebulin. Taken together, the biochemical findings of this study are presented in Fig. 9. Improvement in meat tenderness with post-mortem storage is associated with the proteolysis of key myofibrillar linkage proteins (i.e. nebulin, etc.) and the autolysis of calpains 1 and 3. It is important to determine whether these processes are coupled or not. To test the hypothesis that autolysis of calpains and myofibrillar protein degradation during post-mortem storage are causally linked to meat tenderization manipulations of the rate of tenderization should result in changes in the rate of autolysis of calpains and myofibrillar protein degradation in the same direction. To conclude, circumstantial evidence supporting a role for calpain 3 in meat tenderization was obtained in this study. These include; (1) calpain 3 was associated with the myofibrillar fraction and gradually autolyzed during post-mortem storage, (2) the kinetics of autolysis of calpain 3 was significantly correlated with the kinetics of tenderization (r=0.921, P40.001) and nebulin proteolysis (r=0.930, P40.001) in the LTL. The above
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evidence supporting a role for calpain 3 in post-mortem tenderization of LTL would be more conclusive if activity of calpain 3 could be measured. Immunochemical methods such as Western analysis and the determination of autolysis have been instrumental in the investigation of the role of calpain 2 in meat tenderization (Boehm et al., 1998). Therefore, the fact that no activity data for calpain 3 were included in this study does not jeopardize the findings of the study. Having said that, it is important to restate that Parr et al. (1999) did not find a correlation between variations in porcine LTL tenderness and the abundance of calpain 3. The discrepancy between our results and those of Parr et al. (1999) may be related to methodology or animal species. A question may be raised based on the findings of this study. If autolysis of calpain 3 is indicative of its activation, and calpain 3 proteolyses myofibrillar proteins such as nebulin and desmin why is a considerable drop in detectable calpain 3 (sarcoplasmic ( ffi 60% and myofibrillar ( ffi 40%) between 0 and 1 day post-mortem not reflected in degradation of these proteins? Before providing an explanation, it is important to realize that meat tenderization is a complex biochemical process involving fracturing by the calpain system (at least nine isozymes in skeletal muscle) of a myriad of myofibrillar linkage proteins. In this study, we evaluated a very narrow window of the metabolic pathway of tenderization. The considerable drop in detectable calpain 3 in both sarcoplasmic ( ffi 60%) and myofibrillar ( ffi 40%) fractions between 0 and 1 day postmortem may be related to the degradation of proteins not involved in meat tenderization. Recently, an argument against a role for calpain 3 in meat tenderization was put forward in a conference paper (Koohmaraie et al., 2002) primarily on the basis that inactivation of calpain 3 by genetic mutations is the
Fig. 9. Schematic illustration of the biochemical findings of the study.
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basis of limb girdle muscular dystrophy type 2 A (LGMD2A) in human. Indeed, it is out of the ordinary that the absence of a protease would result in muscle wasting rather than an increase in muscle mass. On this point Tidball and Spencer (2000) provided an explanation for this intriguing phenomenon. They indicated that the above view reflected the expectation that calpain 3’s function was a general proteolytic role in skeletal muscle, rather than a specific influence on regulatory mechanisms. Recent findings have shown that calpain 3 cleaves IkB and that patients with LGMD2A accumulate IkB, which results in myonuclear apoptosis (Baghdiguian et al., 1999). Having said that, it is important to indicate that there is no experimental evidence to indicate that apoptosis has any role in postmortem tenderization. These findings and the observations of increased calpain 3 protein concentration in muscle cachexia (Williams, DeCourten-Myers, & Fischer, 1999) support the view that muscle wasting can result from disruption of normal regulatory events that are mediated by calpain 3, by either a pathological increase or genetic inactivation of calpain 3. Therefore, the potential role of calpain 3 in meat tenderness deserves further study. The calpain 3 mediated degradation of myofibrillar linkage proteins such as nebulin needs to be demonstrated and the relative contribution of calpain 3 proteolysis compared with other degradation pathways must be determined.
Acknowledgements The authors thank Dr. Marion Greaser and Dr. David Hopkins for the valuable comments on the manuscript. We thank the Foundation for Research, Science and Technology (FRST) of New Zealand for funding the work. We extend very special thanks to Dr. Sorimachi for the gift of calpain 3 mutant protein, which was instrumental for the validation of the specificity of our antibody.
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The relationship between meat tenderization, myofibril fragmentation and autolysis of calpain 3 during post-mortem aging Mohammad A. Ilian*, Alaa El-Din Bekhit, Roy Bickerstaffe Molecular Biotechnology Group, Animal and Food Sciences Division, PO Box 84, Lincoln University, Canterbury, New Zealand Received 21 November 2002; received in revised form 4 March 2003; accepted 22 April 2003
Abstract The objective was to study the potential role of calpain 3 in postmortem myofibril breakdown and meat tenderization. We determined the temporal changes in calpain 3 protein in the ovine m. longissimus thoracis et lumborum (LTL, n=4) during postmortem storage. Concurrently, we also determined the kinetics of tenderization level, changes in MFI, degradation of nebulin and desmin, and autolysis of calpain 1. The autolysis of calpains 1 and 3 were strongly correlated with the kinetics of tenderization and changes in MFI. The best correlation was between the appearance of the autolyzed calpains 1 and 3 and nebulin degradation. Taken together, the results indicated that calpains 1 and/ or 3 might be playing a key role in post-mortem tenderization of LTL via the proteolysis of specific muscle structural proteins such as nebulin. This is the first report that relates calpain 3 to myofibrillar protein degradation in post-mortem skeletal muscle. # 2003 Elsevier Ltd. All rights reserved. Keywords: Skeletal muscle; Tenderness; Proteolysis; Calpain 3
1. Introduction Calpains (EC 3.4.22.17) constitute a large family of intracellular Ca2+-dependent cysteine neutral proteinases which are believed to be involved in many Ca2+-regulated physiological as well as pathological conditions (Sorimachi, Ishiura, & Suzuki, 1997; Huang & Wang, 2001). The calpains are classified on the basis of tissue distribution into ubiquitous and tissue-specific. At the last count, the calpain proteolytic system in mammalian skeletal muscle is composed of eight ubiquitous (calpains 1, 2, 5, 7, 10, 12, 14 and 15) and one tissue-specific (calpain 3) isoenzymes. Dayton, Goll, Zeece, Robson, and Reville (1976) purified the first calpain (calpain 2) from porcine skeletal muscle. Since that time, a great deal of experimental evidence has accumulated, indicating that the calpain proteolytic system has a primary role in myofibrillar protein degradation (Barnoy, SupinoRosin & Kosower, 2000; Goll, Thompson, Taylor, &
* Corresponding author. Tel.: +64-3-3253803; fax: +64-3-3253851. E-mail address: [email protected] (M.A. Ilian). 0309-1740/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0309-1740(03)00125-6
Christiansen, 1992; Huang & Forsberg, 1998; Poussard et al., 1996). In post-mortem skeletal muscles of meat animals, the proteolytic activity of the calpain system on the myofibrillar proteins has the primary influence on meat tenderness (Bickerstaffe, 1996; Koohmaraie, 1992; Ouali, 1992). Therefore, tangible benefits could be achieved by understanding the mechanism of meat tenderization. Complete understanding of the biochemical basis of meat tenderization requires the identification of the calpains associated with this process and their protein substrates. Calpains 5, 7, 10, 12, 14 and 15 were recently discovered (Dear & Boehm, 1999; Dear, Meier, Hunn, & Boehm, 2000; Franz, Vingron, Boehm, & Dear, 1999; Horikawa et al., 2000; Kamei, Webb, Young, & Campbell, 1998) and their biochemical role in skeletal muscle and meat is unknown. A recent conference review paper (Geesink, Ilian, Morton, & Bickerstaffe, 2000) on the role of calpains 1, 2 and 3 in meat tenderization concluded the following: ‘‘1) Post-mortem tenderization is primarily caused by m-calpain (calpain 1)-mediated degradation of key muscle structural proteins. 2) Unless meat is treated with mM levels of calcium, m-calpain
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(calpain 2) appears not to be involved in post-mortem tenderization. 3) The role of calpain 3 in tenderization remains to be determined’’. A very recent comprehensive review on current theories of tenderization of red meat concluded that the major candidate to explain tenderization post-rigor is the calpain system and pointed out that among the issues that remain to be resolved is the contribution of calpain 3 to tenderization (Hopkins & Thompson, 2002). The interest in calpain 3 with regard to postmortem tenderization of meat stems from the observations that calpain 3 is a muscle-specific calpain (Sorimachi et al., 1989) and that it binds to titin at the N2 line, a site where proteolysis in the early post-mortem period has been linked to tenderization (Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). The reports on the role of calpain 3 in meat tenderization are very limited due to constraint of investigative tools. With the development of analytical methods for the determination of calpain 3 expression at the mRNA and protein levels some information on the role of calpain 3 in meat tenderization has been published. The first publication on the role of calpain 3 in meat tenderization was presented by Parr et al. (1999) who did not find a correlation between variations in porcine m. longissimus thoracis et lumborum (LTL) tenderness and variations in the level of calpain 3. By comparison, our previous studies on the relationship between calpain 3 and meat tenderization revealed, for the first time, a strong correlation between the variations in ovine meat tenderness and the expressions of calpain 3 at the protein (Ilian et al., 2001a) and mRNA (Ilian et al., 2001b) levels. The objective of this study was to gain insight into the biochemical role of calpain 3 in meat tenderization in comparison with calpain 1. We determined the temporal changes in the protein status of calpain 3 in the ovine LTL during postmortem storage. Concurrently, we also determined the temporal changes of; (1) tenderization level, (2) myofibrillar fragmentation index (MFI), (3) proteolysis of nebulin and desmin, and (4) autolysis of calpain 1. The relationship between calpain 3, meat tenderization and myofibrillar protein degradation during post-mortem storage is discussed on the basis of the results obtained.
2. Materials and methods 2.1. Animals and sample collection Four female Coopworth lambs, age 4.5 months were slaughtered by captive-bolt stunning and exanguinated and dressed at the Lincoln University large animal anatomy laboratory. A sample ( ffi 7g) from the right side LTL was extracted from the L7 vertebra area about 15 min after killing and snap frozen in liquid nitrogen for Western analysis of nebulin, desmin and calpains 1
and 3. The carcasses were kept for 4 h at 15 C and then moved to a 2 C chiller for 7 days. At 1, 2, 3, 4, 5, 6 and 7 days post-mortem, a sample ( ffi 7g) and a steak (about 5 cm5 cm) from the right side LTL were removed from the L6-5, L4-3, L2-1, T13-12, T11-10, T9-7, T6-4 regions, respectively for laboratory analyses. The samples were immediately frozen in liquid nitrogen. The steaks were placed in plastic bags and stored in a freezer at 20 C. 2.2. Tenderness assays Frozen steaks were thawed overnight at 2 C and LTL tenderness was determined subjectively by measuring the force required to shear through a cooked sample as was described earlier (Ilian et al., 2001b). 2.3. Myofibril fragmentation index The myofibril fragmentation index (MFI) was determined on frozen samples taken at 1, 2, 3, 4, 5, 6 and 7 days of post-mortem according to the method described by Culler, Parrish, Smith, and Cross (1978) with some modification. This included using a Polytron1 (Kinemacia, Littau, Switzerland) at 10,000 rpm for 30 s to homogenize the tissue. 2.4. Western blot assays To study the kinetics of the proteolysis of nebulin and desmin and the autolysis of calpains 1 and 3 during post-mortem storage, we separated the LTL proteins from the samples taken after 0, 1, 2, 3, 4, 5, 6 and 7 days of post-mortem storage into sarcoplasmic and myofibrillar fractions. This was performed according to the method described by Boehm, Kendall, Thompson, and Goll (1998) with some modification (Ilian et al., 2001a). Protein concentration of the various fractions was determined according to Karlsson, Ostwald, Kabjorn, and Anderson (1994). Nebulin SDS–PAGE was performed according to Huff-Lonergan, Parrish, and Robson (1995) to allow for the separation of high molecular weight proteins. Other proteins were separated on SDS– PAGE (stacking gel, 3.5% polyacrylamide; separating gel, 8% polyacrylamide) using mini-gels according to Laemmli (1970). Western blot assays of myofibrillar and sarcoplasmic fractions were conducted by loading 27.5 and 9.1 mg protein respectively together with pre-stained broad range SDS–PAGE standards (BIO-RAD, CA). After electrophoresis, gels were electroblotted on polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK) at 4 C overnight and 30 V using a trans-blot unit (BIO-RAD, CA) according to Towbin, Staedhin, and Gordon (1979). The transfer buffer (pH 8.3) was 25 mM Tris, 192 mM glycine, 15% methanol, and 0.5% SDS. Gels
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after transfer were stained with Coomassie Blue to examine the efficiency of transfer. Primary antibodies used in the Western blot assays were as following. (1) Monoclonal anti-nebulin NB2 clone at 1:250 (Sigma Chemical, St. Louis, Mo). (2) Monoclonal anti-desmin Clone D3 at 1:100 (developed by D. A. Fishman, Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242 under the contract N01-HD7-3263 from the NICHD). (3) Monoclonal anti-calpain 1 clone MA3-941 at 1:1000 (Affinity Bioreagents, Golden, CO). (4) Polyclonal anti-calpain 3 Calp3IS2:b/o at 1:500 and a description of the preparation and purification of the Calp3IS2:b/o was given previously (Ilian et al., 2001a). The specificity of all antibodies used in this study for the analyses of their respective antigen was confirmed by producing the respective signals for their antigens on Western blot. The quantitative aspect of the assays of antibodies used in this study for the analyses of their respective antigen were determined by constructing a calibration curve, which showed a linear response of image density to gradation in LTL protein level (results not shown). Primary and secondary antibodies were diluted in blocking buffer (100 mM Tris–Cl, pH 7.5, 0.9% NaCl, 0.1% Tween 20, 5% nonfat milk powder) according to Towbin et al. (1979). After washing, detection of bound antibodies was performed with a chemiluminescence kit (Pierce, IL) and the intensity of signal was determined using a UVP gel documentation system (UVP Inc. Upland, CA). 2.5. Analyses of data Temporal improvements in LTL tenderness, designated here as tenderization level, for each animal (n=4) with time were calculated as a percentage of improvement in tenderness at day 7 post-mortem using shear force value at day 1 as a baseline. The equation for calculating tenderization level was ðkgf 1 kgfn Þ ðkgf 1 kgf 7 Þ 100 where kgfn is shear force value at day n. To determine the effect of time on LTL tenderization level and MFI, data were fitted to an exponential rise to maximum (single variable, three constants) model using the equation Y ¼ Y0 þ a 1 ebX with LTL tenderization level or MFI as the response and time post-mortem as the predictor. Also, one way ANOVA was performed using tenderization level and MFI values as dependent variables and time as the factor level to detect significant differences among the means. To determine the relationship between tenderization level and MFI we performed regression analysis by fitting a linear regression model to the data using the equation Y ¼ 0 þ 1 X with MFI as the response and tenderization level as the predictor. To study the effect of time on the proteolysis of
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nebulin and desmin and the autolysis of calpains 1 and 3 during post-mortem storage the data for each protein from each animal were normalized as percentages of the value at slaughter. Then the normalized values of 1–7 days post-mortem were fitted to an exponential decay (single variable, three constants) model using the equation Y ¼ Y0 þ a ebX with protein level as the response and time post-mortem as the predictor. The exponential regressions provided a means for showing the relationship between myofibril fragmentation and autolysis of calpains 1 and 3. To evaluate the relationship between tenderization level, MFI, proteolysis of nebulin and desmin and autolysis of calpains 1 and 3, the Pearson product moment correlation coefficient between each pair of the above variables was calculated. The software used for performing the above statistics and curve fitting was Minitab version 11 computer package (State College, PA).
3. Results and discussion 3.1. Specificity of anti-calpain 3 antibody Quantitative determination of calpain 3 expression is very important in the study of its function in skeletal muscle and meat. Unlike the ubiquitous calpains 1 and 2, analysis of the activity of calpain 3 is not achievable at the present time because of its propensity to undergo very rapid autolysis during extraction (Kinbara et al., 1998). Having said that, this does not mean that alternative approaches are not available to conduct meaningful research on the role of calpain 3 in meat tenderization. The expression of the calpain proteolytic system at the mRNA (Ilian et al., 2001b; van den Hemel-Grooten, Te Pas, van den Bosh, Garssen, Schreurs, & Versegen, 1997) and protein levels (Ilian et al., 2001a; Parr et al., 1999) have often been determined to understand their role in muscle functioning. In this study, established immunochemical techniques were employed to study the biochemical pathway of meat tenderization during post-mortem storage and the role of calpain 3 in this process. The validity of analysis of calpain 3 in this study absolutely depends on the specificity of the antibody. Details for the development and validation of our anticalpain 3 antibody Calp3IS2:b/o for western assay of bovine and ovine calpain 3 were reported earlier (Ilian et al., 2001a). In brief; 1. We have sequenced the bovine and the ovine calpain 3 cDNA and used the IS2 region, which is unique to calpain 3, for designing the antigenic polypeptide of Calp3IS2:b/o. 2. We have conducted a BLAST search to examine the homology of the antigenic polypeptide of
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Calp3IS2:b/o to other proteins. The results indicated that the antigenic peptide is unique to calpain 3 and not present in other known proteins. 3. Based on published results, calpain 3 is a skeletal muscle specific enzyme with 94 kDa molecular weight (Sorimachi et al., 1989). We utilized this information to examine the specificity of Calp3IS2:b/o. We tested Calp3IS2:b/o with proteins from skeletal muscle, liver, heart and lung. Calp3IS2:b/o only reacted with proteins from skeletal muscle and produced a signal at 94 kDa (Ilian et al., 2001a). 4. Furthermore, we tested the specificity of Calp3IS2:b/o using pure p94:C129S. Protein p94:C129S is a calpain 3 mutant in which the active site (Cys-129) had been changed to serine to eliminate the protease activity (Kinbara et al., 1998). Calp3IS2:b/o reacted with p94:C129S very strongly. Essentially the same signal was obtained with the skeletal muscle (Ilian et al., 2001a). 5. During the preparation of the manuscript, Koohmaraie, Kent, Shackelford, Veiseth, and Wheeler (2002) described unpublished work from Goll laboratory indicating their calpain 3 antibodies reacted with 94 kDa non-calpain proteins (determined by micro-sequencing). Therefore the Calp3IS2:b/o antibody was tested against two prominent muscle proteins of this approximate size, phosphorylase and phosphofructokinase (PFK). A blot using purified phosphorylase a and b, PFK, (Sigma Australia) plus skeletal muscle and the p94:C129S protein as positive controls is shown in Fig. 1. The results indicated that Calp3IS2:b/o labeled p94:C129S and skeletal muscle but did not label phosphorylases a and b or PFK even at the high concentration used (200 ng). The explanation for the faint trail in the p94:C129S lane of Fig. 1 is that during the purification of this mutant inactive calpain 3,
Fig. 1. SDS–PAGE of skeletal muscle phosphorylase a and b, skeletal muscle phosphofructokinase, p94:C129S pure proteins together with whole ovine longissimus proteins and Western analysis of same using Calp3IS2:b/o.
the proteolytic enzymes of the COS cells caused limited degradation of the p94:C129S (Sorimachi, personal communication). No implications of using p94:C129S for the validation of the specificity of Calp3IS2:b/o for the detection of native calpain 3 are anticipated. This is because Calp3IS2:b/o was raised against an epitope in the IS2 region, which is distant from the active site region.
3.2. Tenderization and MFI profiles The kinetics of tenderization of LTL (expressed as percentage of the tenderization at 7 days) over a period of 7 days post-mortem storage under refrigerated conditions are presented in Fig. 2a. Presentation of shear
Fig. 2. Effect of postmortem storage period on tenderness and myofibrillar fragmentation index in ovine m. longissimus thoracis et lumborum (LTL). Kinetics of tenderization and myofibrillar fragmentation index of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions are presented in panels a and b, respectively. Regression analysis between the results of tenderization level and MFI during 1–7 days post-mortem is shown in panel c.
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force values as tenderization level was done for the following reasons. (1) To normalize the results across the experimental animals used in the study. (2) To provide a clearer relationship between changes in LTL tenderness and time of post-mortem storage. (3) To provide a direct, not inverse, mathematical relationship between tenderness and myofibrillar fragmentation index. The results indicate that significant improvements in LTL tenderness occurred gradually during the first three days of post-mortem storage. On average, 44 and 72% improvements over the 1 days tenderness values were observed at days 2 and 3 post-mortem, respectively. This is in agreement with previous results reported by our laboratory (Ilian et al., 2001b) and others (Wheeler & Koohmaraie, 1994). To gain insight into whether tenderization of LTL is primarily due to myofibrillar protein degradation, we determined the kinetics of MFI over a period of 7 days post-mortem storage under refrigerated conditions as shown in Fig. 2b. MFI is a useful indicator of the extent of myofibrillar protein degradation (Olson, Parrish, & Stromer, 1976). Significant increases in MFI occurred gradually during days 2 and 3 of post-mortem storage compared with day 1. To determine the relationship between tenderization level and MFI we fitted a linear regression model to the data using the equation Y ¼ 0 þ 1 X with MFI as the response and tenderization level as the predictor (Fig. 2c). The results revealed a strong correlation (r=0.787, P40.01) between the kinetics of tenderization and MFI indicating that myofibrillar protein degradation during post-mortem storage of LTL was closely associated with tenderization in this study.
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Fig. 3. Kinetics of nebulin proteolysis in the myofibrillar fraction of ovine LTL (n=4) during 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of nebulin proteolysis.
3.3. Proteolysis of nebulin and desmin As a confirmation for the MFI results we examined myofibrillar protein degradation in LTL during postmortem storage at the molecular level. Western blot assays were used to measure the kinetics of proteolysis of nebulin and desmin in LTL over a period of 7 days post-mortem storage under refrigerated conditions (Figs. 3 and 4). Nebulin and desmin were investigated for the following reasons. Firstly, they play important roles in organizing and maintaining the integrity and strength of the contractile myofibrils and the overall cytoskeleton structure of the skeletal muscle cell (Robson et al., 1997). Nebulin and desmin are also known to undergo post-mortem proteolysis at a fast and slow rate respectively (Robson et al., 1997). Finally, they are excellent substrates for the calpain proteolytic system (Huff-Lonergan, Mitsuhashi, Beekman, Parrish, Olson, & Robson, 1996). Kinetics of nebulin proteolysis in LTL (as percentage of value at slaughter) over a period of 1–7 days postmortem storage at 2 C is presented in Fig. 3. Essentially no fragmentation of nebulin occurred during the
Fig. 4. Kinetics of desmin proteolysis in the myofibrillar fraction of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of desmin proteolysis.
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first 24 h post-mortem. However, complete fragmentation of nebulin occurred by 3 days post-mortem. After 2 and 3 days post-mortem, about 70 and 97% of intact nebulin was degraded, respectively. The kinetics of desmin proteolysis in LTL over a period of 1–7 days post-mortem storage at 2 C is presented in Fig. 4. As with nebulin, very limited degradation of desmin occurred during the first 24 h post-mortem. The rate of degradation of desmin, however, is much slower than that of nebulin. After 2 days post-mortem, some desmin was degraded but the results were not significantly different from the level at slaughter. Significant amounts of desmin were degraded after 3 and 5 days post-mortem storage with losses of 33 and 71% of intact desmin, respectively. Therefore, the above results confirm the results of MFI and indicate that the biochemical basis of LTL tenderization during post-mortem aging in this study is the breakdown of certain myofibrillar proteins including nebulin and desmin. However, the rate of degradation, in agreement with others (Robson et al., 1997), depends upon storage time and protein species. 3.4. Autolysis of calpains It is believed that the calpain proteolytic system has more than one mechanism of activation depending on the cellular condition and that autolysis is perhaps one of the mechanisms of calpain activation (Molinari, Anagli, & Carafoli, 1994; Saido, Suzuki, Yamazaki, Tanoue, & Suzuki, 1993; Suzuki, Tsuji, Kubota, Kimura, & Imahori, 1981). Several aspects of activity regulation of calpains in relation to autolysis have been investigated. These include; the effect of autolysis on Ca2+ sensitivity (Baki, Tompa, Alexa, Molnar, & Friedrich, 1996; Goll, Thompson, Taylor, & Zalewska, 1992), substrate specificity (Schoenwaelder et al., 1997), subunits interaction and proteolytic activity (Suzuki & Sorimachi, 1998). It is important to indicate that autolysis of calpains was observed in pathological conditions associated with aberrant rise in the concentration of intracellular calcium (Vanderklish & Bahr, 2000). The most clearly documented biological relevance of calpain autolysis in the literature does not involve a function in living cells, but rather its role in postmortem tenderization of meat. Koohmaraie, Seideman, Schollmeyer, Dutson, and Crouse, (1987) proposed that calpain 1, and not calpain 2, is responsible for meat tenderization because its activity gradually declined during post-mortem aging of meat. Boehm et al. (1998) conducted a rigorous study on the changes in activity and protein structure of calpains 1 and 2 in bovine skeletal muscle during the first 7 days of post-mortem storage. They determined that extractable calpain 2 activity decreased slightly during the first 7 days post-mortem however, calpain 1 activity decreased to less than 4% of
at-death value. Western blot and sequencing analyses showed that the 80 kDa subunit of calpain 2 remained intact during the first 7 days postmortem however the 80 kDa subunit of calpain 1 was almost entirely converted to the 76 kDa autolyzed form. The authors concluded that calpain 2 is not proteolytically active during post-mortem storage of meat because it was not autolyzed. In a recent study, Veiseth, Shackelford, Wheeler, and Koohmaraie, (2001) utilized casein zymography to study the autolysis of calpains 1 and 2 in ovine meat during postmortem storage. Their results revealed that only calpain 1 showed autolysis under post-mortem conditions. A conservative interpretation of the above data is that autolysis indicates that a calpain has been activated, although subsequent modifications may cause inactivation. Thus, autolysis can be used as an assay of activation, but not activity (Tidball & Spencer, 2000). In this study, we determined the effect of post-mortem storage on the protein status of calpains 1 and 3. Calpain 1 was used for comparative purposes. An important aim here was to determine the association of calpain 3, like calpain 1, with LTL myofibrillar proteins. The underlying hypothesis was that in order for a protease to be involved in meat tenderization it must have access to the substrate and be associated with the myofibril. The myofibrillar-bound calpain 1 has been the subject of recent publications (Boehm et al., 1998; Delgado, Geesink, Marchello, Goll, & Koohmaraie, 2001). Western blot analyses of the large subunit of calpain 1 in the sarcoplasmic and myofibrillar fractions of LTL during a period of 7 days post-mortem storage are presented in Figs. 5 and 6, respectively. The results indicate that the catalytic subunit of calpain 1 is present in both the sarcoplasmic and myofibrillar fractions at slaughter primarily in the form of the 80 kDa form. Subsequently, the calpain 1 native catalytic subunit in both the sarcoplasmic and myofibrillar fractions was gradually autolyzed in the LTL during post-mortem storage. The autolysis kinetics of sarcoplasmic calpain 1 catalytic subunit, measured as the abundance of the 80 kDa subunit, indicate that the enzyme was autolyzed after slaughter and lost about 50 and 85% of its native form by 1 and 2 days post-mortem, respectively (Fig. 5). In contrast, the concentration of myofibrillar calpain 1 native catalytic subunit was not changed at 1 day postmortem compared with the initial value at slaughter (Fig. 6). Subsequently, myofibrillar calpain 1 native catalytic subunit autolysis was steep as 75% of the 80 kDa catalytic subunit was autolysed by 2 days postmortem. The autolysis pattern and localization of the native sarcoplasmic and myofibrillar calpain 1 catalytic subunit may indicate different functions. Logically, myofibrillar calpain 1 may be the one involved in myofibrillar protein degradation during post-mortem aging because of its association with the myofibrils.
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Fig. 5. Kinetics of autolysis of the catalytic subunit of calpain 1 in the sarcoplasmic fraction of ovine LTL (n=4) during a 7 days postmortem storage period under refrigerated conditions. Upper panel is a representative Western blot of calpain 1 catalytic subunit in the sarcoplasmic fraction.
Fig. 6. Kinetics of autolysis of the catalytic subunit of calpain 1 in the myofibrillar fraction of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of calpain 1 catalytic subunit in the myofibrillar fraction.
The propensity of calpain 3 to autolysis has been clearly documented (Kinbara et al., 1998). Early literature indicated that calpain 3 is destroyed by autolysis with a half-life of approximately 30 min in COS cells (Sorimachi et al., 1993). Recent studies, however, have shown that the above phenomenon is applicable to calpain 3 in skeletal muscle tissue but with much slower rates. For example, calpain 3 protein was found to be stable in fresh human muscle, with full-size protein being detected 8 h after the muscle had been removed (Anderson et al., 1998). Jones, Parr, Sensky, Scothern, Bardsley, and Buttery (1999) explained that calpain 3 autolyzes rapidly when expressed in COS cells, however, in vivo the enzyme may be stabilized by interaction with titin. Last but not least, intact calpain 3 protein in porcine longissimus muscle could be detected up to 24 h after slaughter in carcasses held in a chiller (Parr et al., 1999). In a previous study, we showed autolysis of calpain 3 in post-mortem skeletal muscle during aging (Ilian et al., 2001a). The autolytic process of calpain 3 in ovine skeletal muscle during post-mortem aging was characterized by a gradual increase in the concentration of a 52.5 kDa autolytic fragment in association with a corresponding gradual decrease of the level of intact 94 kDa protein. Unlike calpains 1 and 2, the kinetics of
autolyzis of calpain 3 and its physiological significance during post-mortem aging of meat has not yet been established. The observations that the three autolysis sites in calpain 3 are present in the catalytic domain II (Kinbara et al., 1998) and that mutations in the autolysis sites were reported in limb girdle muscular dystrophy type 2A patients (Beckmann et al., 1996) are indications that autolysis may be an important aspect of a functional calpain 3. Furthermore, Kinbara and associates (1998) indicated that the ffi 55 kDa fragment (in our study 52.5 kDa) is the C-terminus fragment of calpain 3 which comprises domain III and VI. Similar information on the nature of the ffi 55 kDa fragment of calpain 3 was given by Branca, Gugliucci, Bano, Brini, and Carafolli, (1999). It is important to indicate that the active site of calpain 3 is located in domain II (Sorimachi et al., 1989) and therefore, the 52.5 kDa fragment is expected to possess no proteolytic activity. In this study, the results of western blot analysis indicated that calpain 3 was present in the sarcoplasmic and myofibrillar fractions at slaughter and that calpain 3 in both fractions expressed autolysis in LTL during postmortem storage (Figs. 7 and 8). The kinetics of autolysis of sarcoplasmic calpain 3 indicate that the enzyme was terminally activated after slaughter and that about 40
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Fig. 7. Kinetics of autolysis of calpain 3 in the sarcoplasmic fraction of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of calpain 3 autolysis in the sarcoplasmic fraction.
and 85% of the native calpain 3 level was lost by 1 and 2 days post-mortem, respectively (Fig. 7). Similarly, the myofibrillar calpain 3 was autolyzed, but at a lower rate, after slaughter and about 25 and 75% of the 94 kDa protein was lost by 1 and 2 days post-mortem, respectively. Afterward, calpain 3 autolysis in the sarcoplasmic and myofibrillar fractions continued, but at a much lower rate, until 5 days post-mortem. 3.5. Calpains and post-mortem meat tenderization It is well established that post-mortem meat tenderization is a complex biochemical process involving the fracturing of key myofibrillar proteins which are responsible for maintaining the structural integrity of the myofibrils by the calpain proteolytic system (Hopkins & Thompson, 2002). However, the mode of action of the calpains in this process is not fully understood. The relative contribution of the various calpains in postmortem tenderization of meat is an open question (Boehm et al., 1998). Detailed understanding of the biochemical role of the calpain proteolytic system in meat tenderization requires the identification of the calpains associated with this process and their protein substrates. Previously, we have examined the role of
Fig. 8. Kinetics of autolysis of calpain 3 in the myofibrillar fraction of ovine LTL (n=4) during a 7 days post-mortem storage period under refrigerated conditions. Upper panel is a representative Western blot of calpain 3 autolysis in the sarcoplasmic fraction.
calpain 3 in meat tenderization using three biological systems. The results, for the first time, have indicated that variations in the expression of calpain 3 at the mRNA and protein levels are strongly correlated to variations in the tenderness of post-mortem skeletal muscle (Ilian et al., 2001a, 2001b). Also the variations in the rate of titin proteolysis due to stretching is directly related to the rate of calpain 3 autolysis (Stevenson, Morton, Ilian, & Bickerstaffe, 2002). To gain insight into the mode of action of calpain 3 in post-mortem meat tenderization we performed correlation analyses for the temporal changes in tenderization level, nebulin and desmin proteolysis and autolysis of calpains 1 and 3 during 1–7 days of post-mortem storage (Table 1). The results indicate that kinetics of tenderization level is Table 1 Correlation matrix for temporal changes in tenderness level, nebulin and desmin proteolysis and calpains 1 and 3 autolysis during 7 days postmortem storage Tenderness Nebulin Desmin Calpain level proteolysis proteolysis 1(M)a autolysis Nebulin proteolysis Desmin proteolysis Calpain 1(M) autolysis Calpain 3(M) autolysis a
M, myofibrillar.
0.932 0.511 0.986 0.921
0.634 0.956 0.930
0.610 0.654
0.986
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highly correlated with the kinetics of nebulin proteolysis (r=0.932, P40.001) and the kinetics of myofibrillar calpains 1 (r=0.986, P40.001) and 3 (r=0.921, P40.001) autolysis. The correlation coefficient between the kinetics of tenderization level and desmin proteolysis was not significant (r=0.511, P40.05). However, this result does not discard a role for desmin proteolysis in the development of tenderness during post-mortem storage. These results suggest that nebulin proteolysis may be a key component in tenderization of meat. Furthermore, the kinetics of nebulin proteolysis was strongly correlated with the autolysis of the catalytic subunit of the myofibrillar calpain 1 (r=0.956, P40.001) and myofibrillar calpain 3 (r=0.930, P40.001). These results provide indirect evidence that calpains 1 and/or 3 may be responsible for the fragmentation of nebulin. Taken together, the biochemical findings of this study are presented in Fig. 9. Improvement in meat tenderness with post-mortem storage is associated with the proteolysis of key myofibrillar linkage proteins (i.e. nebulin, etc.) and the autolysis of calpains 1 and 3. It is important to determine whether these processes are coupled or not. To test the hypothesis that autolysis of calpains and myofibrillar protein degradation during post-mortem storage are causally linked to meat tenderization manipulations of the rate of tenderization should result in changes in the rate of autolysis of calpains and myofibrillar protein degradation in the same direction. To conclude, circumstantial evidence supporting a role for calpain 3 in meat tenderization was obtained in this study. These include; (1) calpain 3 was associated with the myofibrillar fraction and gradually autolyzed during post-mortem storage, (2) the kinetics of autolysis of calpain 3 was significantly correlated with the kinetics of tenderization (r=0.921, P40.001) and nebulin proteolysis (r=0.930, P40.001) in the LTL. The above
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evidence supporting a role for calpain 3 in post-mortem tenderization of LTL would be more conclusive if activity of calpain 3 could be measured. Immunochemical methods such as Western analysis and the determination of autolysis have been instrumental in the investigation of the role of calpain 2 in meat tenderization (Boehm et al., 1998). Therefore, the fact that no activity data for calpain 3 were included in this study does not jeopardize the findings of the study. Having said that, it is important to restate that Parr et al. (1999) did not find a correlation between variations in porcine LTL tenderness and the abundance of calpain 3. The discrepancy between our results and those of Parr et al. (1999) may be related to methodology or animal species. A question may be raised based on the findings of this study. If autolysis of calpain 3 is indicative of its activation, and calpain 3 proteolyses myofibrillar proteins such as nebulin and desmin why is a considerable drop in detectable calpain 3 (sarcoplasmic ( ffi 60% and myofibrillar ( ffi 40%) between 0 and 1 day post-mortem not reflected in degradation of these proteins? Before providing an explanation, it is important to realize that meat tenderization is a complex biochemical process involving fracturing by the calpain system (at least nine isozymes in skeletal muscle) of a myriad of myofibrillar linkage proteins. In this study, we evaluated a very narrow window of the metabolic pathway of tenderization. The considerable drop in detectable calpain 3 in both sarcoplasmic ( ffi 60%) and myofibrillar ( ffi 40%) fractions between 0 and 1 day postmortem may be related to the degradation of proteins not involved in meat tenderization. Recently, an argument against a role for calpain 3 in meat tenderization was put forward in a conference paper (Koohmaraie et al., 2002) primarily on the basis that inactivation of calpain 3 by genetic mutations is the
Fig. 9. Schematic illustration of the biochemical findings of the study.
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basis of limb girdle muscular dystrophy type 2 A (LGMD2A) in human. Indeed, it is out of the ordinary that the absence of a protease would result in muscle wasting rather than an increase in muscle mass. On this point Tidball and Spencer (2000) provided an explanation for this intriguing phenomenon. They indicated that the above view reflected the expectation that calpain 3’s function was a general proteolytic role in skeletal muscle, rather than a specific influence on regulatory mechanisms. Recent findings have shown that calpain 3 cleaves IkB and that patients with LGMD2A accumulate IkB, which results in myonuclear apoptosis (Baghdiguian et al., 1999). Having said that, it is important to indicate that there is no experimental evidence to indicate that apoptosis has any role in postmortem tenderization. These findings and the observations of increased calpain 3 protein concentration in muscle cachexia (Williams, DeCourten-Myers, & Fischer, 1999) support the view that muscle wasting can result from disruption of normal regulatory events that are mediated by calpain 3, by either a pathological increase or genetic inactivation of calpain 3. Therefore, the potential role of calpain 3 in meat tenderness deserves further study. The calpain 3 mediated degradation of myofibrillar linkage proteins such as nebulin needs to be demonstrated and the relative contribution of calpain 3 proteolysis compared with other degradation pathways must be determined.
Acknowledgements The authors thank Dr. Marion Greaser and Dr. David Hopkins for the valuable comments on the manuscript. We thank the Foundation for Research, Science and Technology (FRST) of New Zealand for funding the work. We extend very special thanks to Dr. Sorimachi for the gift of calpain 3 mutant protein, which was instrumental for the validation of the specificity of our antibody.
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