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μ-Calpain is involved in the postmortem proteolysis of gizzard smooth muscle
Food Chemistry 139 (2013) 384–388
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
l-Calpain is involved in the postmortem proteolysis of gizzard smooth muscle Ya-Shiou Chang a, Marvin H. Stromer b, Rong-Ghi R. Chou a,⇑ a b
Department of Animal Science, National Chiayi University, Chiayi City, Taiwan Department of Animal Science, Iowa State University, Ames, IA, USA
a r t i c l e
i n f o
Article history: Received 14 August 2012 Received in revised form 9 January 2013 Accepted 28 January 2013 Available online 9 February 2013 Keywords: Gizzard smooth muscle Postmortem proteolysis Calpain Desmin
a b s t r a c t Postmortem changes in proteins that have been implicated in affecting muscle integrity were examined in goose (GG) and duck (DG) gizzard smooth muscle stored at 5 °C. GG and DG smooth muscles were sampled at 0, 1, 3 and 7 day of storage. The pH was approximately 7 in both GG and DG samples during postmortem storage. Casein zymograms showed that 0-day l-calpain activity was higher (p < 0.05) in GG than in DG samples. As postmortem time progressed, l-calpain was activated and autolyzed more extensively in GG than in DG samples. However, l/m-calpain remained relatively stable in both samples. Western blots indicated that postmortem desmin degradation was more rapid in GG than in DG samples. In contrast, a-actinin remained nearly unchanged in both samples. Therefore, our results suggest that l-calpain has an important role in the postmortem proteolysis of gizzard smooth muscle. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Postmortem changes in skeletal muscle stored at 0–5 °C have been extensively studied. The most notable of the changes is the degradation of myofibrillar/cytoskeletal proteins. Ouali et al. (2006) proposed that this proteolysis might involve a multi-enzymatic process. Among the endogenous protease systems in skeletal muscle, evidence has shown that the calpains are able to access their myofibrillar/cytoskeletal substrates and to reproduce the proteolysis pattern observed during postmortem storage of skeletal muscle (Koohmaraie & Geesink, 2006). By fulfilling those criteria, the calpains, especially l-calpain, appear to play an essential role in the postmortem proteolysis of skeletal muscle (Geesink, Kunchay, Chishti, & Koohmaraie, 2006). However, the possible role of other endogenous proteases such as the lysosomal cathepsins (Uytterhaegen, Claeys, & Demeyer, 1994), the proteasome (Houbak, Ertbjerg, & Therkildsen, 2008) and caspases (Kemp, King, Shackelford, Wheeler, & Koohmaraie, 2009; Mohrhauser, Underwood, & Weaver, 2011) have also been implicated. Poultry gizzard is not only a by-product in the poultry industry, but is also a popular food item in oriental cuisine and traditional meat snacks in Asia. The gizzard is mainly composed of smooth muscle and connective tissue (Ruantrakool & Chen, 1986). It was reported that connective tissue (or collagen) did not undergo significant changes during postmortem storage at 0–5 °C (McCormick, 1994). However, little information is available regarding postmor-
tem proteolysis in gizzard smooth muscle stored at 0–5 °C. The purpose of this study, therefore, was to examine whether the calpains were involved in the postmortem proteolysis of goose and duck gizzard smooth muscles stored at 5 °C, and also to compare the differences in postmortem proteolysis between goose and duck gizzard smooth muscles. 2. Materials and methods 2.1. Sample preparation The Institutional Animal Care and Use Committee, National Chiayi University, approved the use of the animals in this study. Gizzards from six White Roman geese and six Pekin ducks were obtained 20 min postmortem from a local slaughter house. Each gizzard was used as one replication. The weight of individual goose and duck gizzards were 140–155 and 115–130 g, respectively. After trimming off any visible connective tissue, each gizzard was immediately cut into four equal portions and allocated to four sampling times. The portions were then vacuum-packed individually and stored at 5 °C for 0, 1, 3, and 7 day. At the end of each storage period, the samples were finely cut with a scalpel and stored in liquid nitrogen until required for subsequent analysis. Duplicate samples from each gizzard, at each storage time, were used for each analysis. 2.2. pH measurement
⇑ Corresponding author. Address: Department of Animal Science, National Chiayi University, 300 University Road, Chiayi City 60083, Taiwan. Tel.: +886 5 2717533; fax: +886 5 2753459. E-mail address: [email protected] (Rong-Ghi R. Chou). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.01.075
A 5 g aliquot from each gizzard sample was crushed in liquid nitrogen for the pH determination by the method of Farouk and Swan (1997). The pH for each sample was measured by a Suntex
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2.3. Casein zymography The procedure used for protein extraction was based on the method of Veiseth, Shackelford, Wheeler, and Koohmaraie (2001). Briefly, a 5 g sample was homogenized in three volumes of extraction buffer (100 mM Tris base, 10 mM EDTA, 0.05% 2mercaptoethanol, pH 8.3) at 5 °C. Homogenates were centrifuged at 22,000 g for 25 min at 5 °C. The protein concentration of the supernatant was determined by using a modified biuret method (Robson, Goll, & Temple, 1968). Zymograms were routinely run in 12% gels (acrylamide:methylenebisacrylamide = 37.5:1, w/w) containing 0.21% casein (w/v) by the method of Raser, Posner, and Wang (1995). The sample buffer (150 mM Tris–HCl, pH 6.8, 20% glycerol, 0.05% 2-mercaptoethanol (MCE), 0.02% [w/v] bromophenol blue) was added to the protein extract at a ratio of 2 parts of the buffer to 3 parts of protein extract (v/v). The same amount of protein (250 lg) from each sample was loaded onto each well of the casein gels. The casein minigels (0.75 mm, Bio-Rad Laboratories, Herculues, CA, USA) were prerun at 100 V for 15 min, 5 °C, with a running buffer containing 25 mM Tris–HCl, 0.05% MCE, 192 mM glycine, and 1 mM EDTA (pH 8.3) before samples were loaded onto the wells. The gels were run at 100 V for 2 h, 5 °C, and then incubated at room temperature in three changes of a 50 mM Tris–HCl (pH 7.5) buffer containing 0.05% MCE and CaCl2 (0.01, 0.03, 0.1 or 4 mM) with slow shaking for 1 h. This was followed by a 16 h incubation in the same buffer at 37 °C. The gels were then stained for 2 h with Coomassie blue (R-250) and destained with 20% methanol and 7% acetic acid. 2.4. Western blot analysis The muscle samples for SDS–PAGE were prepared by the method of Fritz and Greaser (1991). Briefly, muscle samples (0.2 g) were crushed in liquid nitrogen and vigorously mixed with the SDS– PAGE sample buffer (4 ml) containing 8 M urea, 2 M thiourea, 3% SDS, 75 mM dithiothreitol, 0.05% bromophenol blue, and 25 mM Tris–HCl, pH 6.8. The mixture was heated at 100 °C for 3 min and sonicated for 10 min at room temperature. The SDS–PAGE was done in a 10% tris–glycine slab gel (acrylamide:methylenebisacrylamide = 37.5:1, w/w). Equal volume of each sample (20 ll) were loaded onto each well. Proteins were transferred from the 10% slab gel to a nitrocellulose membrane. After the transfer, the membrane was incubated in a 5% bovine serum albumin–phosphate buffer solution (BSA–PBS) for 30 min at 37 °C and was then washed, three times (5 min each), in a 0.1% BSA–PBS solution at room temperature. A monoclonal antibody to desmin (Clone DE-U-10, 1:500 dilution) or a-actinin (Clone BM-75.2, 1:300 dilution) was used as a primary antibody. The membrane was incubated with the primary antibody for 2 h at 25 °C, washed three times (5 min each) in 0.1% BSA–PBS, incubated with a secondary antibody, goat anti-mouseHRP for 2 h at room temperature, washed twice (5 min each) in 0.1% BSA–PBS solution and twice (1 min each) in deionized water. The colour was developed by using SIGMAFASTTM 3,30 -diaminobenzidine tablets. The primary and secondary antibodies, as well as the SIGMAFASTTM 3,30 -diaminobenzidine tablets, were purchased from the Sigma–Aldrich Company (St. Louis, MO, USA).
J131B, Epson), using Photoshop software. Each blot or gel included a pooled 0 day GG sample as a reference standard to normalize the band intensities. The resulting signals were quantified using Image Gauge (version 3.46, Science Lab 99 for Windows, Fuji Film, Tokyo, Japan). The relative l-calpain activity, as well as the relative l/mcalpain activity of 1, 3, and 7 day postmortem GG samples and of 0, 1, 3, and 7 day postmortem DG samples was expressed as a percentage of the relative l-calpain activity and l/m-calpain activity present in the 0 day GG samples. On the other hand, the relative desmin and a-actinin contents in the 0 day GG and DG samples were taken as 100% with respect to each gizzard sample. 2.6. Statistical analysis Split-plot design was used in this study. Whole units were the gizzards from each avian species, and subunits were the smooth muscle samples taken at each sampling time. All data were analyzed using the Mixed Model procedure of SAS (PROC Mixed) (SAS Institute Inc., 2004). The model included avian species, time postmortem and their interaction (species time postmortem) for fixed effects and birds for random effects. A Tukey’s honestly significant difference test was used to compare multiple means. 3. Results and discussion 3.1. Postmortem changes in smooth muscle pH The changes in postmortem pH of goose (GG) and duck (DG) gizzard smooth muscles are shown in Fig. 1. The results showed that the pH was insignificantly different (p > 0.05) between GG and DG samples during a 7 day postmortem storage at 5 °C. The average 0 day pH was 7.08 ± 0.03 in GG samples and was 7.10 ± 0.11 in DG samples and remained nearly unchanged (p > 0.05) in both samples throughout the 7 day sampling period. These results indicated that the ultimate pH in both gizzard smooth muscles was close to 7, which was higher than goose cardiac (6.12) and breast (5.96) muscles (Ho, Lin, & Chou, 2008). It was reported that the glycogen content of gizzard smooth muscle was approximately nine times and two and half times lower than that of skeletal muscle and of cardiac muscle, respectively (Gröschel-Stewart & Zuber, 1990). This suggested that lactic acid production from postmortem glycolysis was limited in gizzard smooth muscle, resulting in a higher ultimate pH. 3.2. Analysis of casein zymography 3.2.1. Activity of l-l/m-, and m-calpain at 0 day Casein zymograms of 0 day GG and DG samples (Fig. 2) showed that one band, with slow migration, clearly appeared in the presence of 10 lM Ca2+ (Fig. 2A). When Ca2+ concentration was 7.5
pH
470 pH meter (Suntex Instruments Co., Taiwan) with a glass electrode.
7.0
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2.5. Image analyses
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Postmortem time, day Either two or three representative casein gels or blots from each gizzard were used for image analysis. The bands in blots or casein gels, which were incubated in the presence of 4 mM Ca2+ to activate all calpain isoforms, were digitized with a scanner (Model
Fig. 1. Postmortem changes in pH of goose (GG) and duck (DG) gizzard smooth muscles stored at 5 °C. Each point (mean ± SD) is the average of six separate replications (n = 6). Vertical bars show the standard deviation of the means. j: GG; h: DG.
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Fig. 2. Zymograms showing l-, l/m-, and m-calpains in the presence of 10 lM (A), 30 lM (B), 100 lM (C), 4 mM (D) calcium, and image analysis results of l- and l/mcalpain activities in the presence of 4 mM calcium in goose (GG) and duck (DG) gizzard smooth muscle 0 day samples (E). Lane 1, GG samples; lane 2, DG samples. l = lcalpain; l/m = l/m-calpain; m = m-calpain. The 0 day GG samples were taken as 100%. Vertical bars show the standard deviation of the means. Different letters within each calpain isoform indicate significant differences (p < 0.05). j: GG; h: DG.
increased to 30 lM, one additional band with an electrophoretic mobility greater than that of l-calpain began to appear (Fig. 2B). These results suggested that the calpain sensitive to 10 lM Ca2+ was l-calpain, and that the calpain with a greater mobility was the l/m-calpain defined by (Sorimachi et al., 1995). Furthermore, no extra bands were found in GG and DG samples in the presence of 100 lM Ca2+ (Fig. 2C). However, an additional band of activity between the l-calpain and the l/m-calpain bands was present in GG, but not in DG, samples in the presence of 4 mM Ca2+ (Fig. 2D). A third calpain, with half-maximal activity at 3.8 mM Ca2+, was purified from mature egg-laying hen skeletal muscle by Wolfe et al. (1989) and might be the skeletal muscle analogue of the additional band we observed in smooth muscle. Sorimachi et al. (1995) proposed that this third calpain might be m-calpain. Therefore, our results imply that, in the presence of 4 mM Ca2+, the additional calpain activity seen in the GG samples might be due to a putative m-calpain. However, this putative m-calpain was absent in the DG samples (Fig. 2D) and in 100 day-old chicken skeletal muscle (Chang & Chou, 2010). Collectively, these results suggested that the presence of this putative m-calpain might depend on avian species and/or animal age. Image analysis of the 0 day zymograms incubated with 4 mM Ca2+ showed that the 0 day activity of l-calpain was 62% higher (p < 0.05) in GG than in DG samples (Fig. 2E). However, no significant difference (p > 0.05) in the 0 day activity of l/m-calpain was found between GG and DG samples (Fig. 2E).
3.2.2. Postmortem changes in l- and l/m-calpain activities Postmortem changes in l- and l/m-calpain activities in GG and DG samples are shown in Fig. 3. The results indicated that l-calpain activity, rather than l/m-calpain activity, appeared to decrease more rapidly in GG than in DG samples as postmortem time progressed (Fig. 3). When the zymograms in Fig. 3 were subjected to image analysis (Fig. 4), the GG l-calpain activity in 1 day
Fig. 3. Zymograms showing postmortem changes in l- and l/m-calpain activities in the presence of 4 mM calcium in goose (A) and duck (B) gizzard smooth muscle samples stored at 5 °C. Lane 1, 0 day samples; lane 2, 1 day samples; lane 3, 3 day samples; lane 4, 7 day samples. l- = l-calpain; l/m- = l/m-calpain; m- = mcalpain.
samples decreased (p < 0.05) to 82% of the 0 day GG l-calpain activity. The level of l-calpain activity continued to decrease (p < 0.05) to 29% and 20% in 3 and 7 day samples, respectively. In contrast, the decrease in l-calpain activity in DG samples was fairly slow (Fig. 4). The DG l-calpain activities were not different (p > 0.05) among the sampling times which were 35% of the 0 day GG l-calpain activity at 0 day, 30% at 1 day, and 27% at 3 day (Fig. 4). The level of l-calpain activity in 7 day DG samples (18% of the 0-day GG l-calpain activity) was below (p < 0.05) from that in 0 day DG samples, but was not different (p > 0.05) from that in 1 and 3 day DG samples. These results indicated that l-calpain activation and autolysis were more rapid and extensive in GG samples than in DG samples. On the other hand, l/m-calpain activity in GG and DG samples remained nearly unchanged (p > 0.05) during the entire 7 day postmortem storage period (Fig. 5).
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Relative activity, %
120 100 80 60 40 20 0
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Fig. 4. Postmortem changes in l-calpain activity of goose (GG) and duck (DG) gizzard smooth muscle samples stored at 5 °C. Activity is expressed as percentage of the 0 day GG l-calpain activity, which was taken as 100%. Vertical bars show the standard deviation of the means. j: GG; h: DG.
Relative activity, %
120 100 80 Fig. 6. Western blot showing changes in desmin and a-actinin of goose (GG) and duck (DG) gizzard smooth muscle samples during postmortem storage at 5 °C. Lane 1, 0 day samples; lane 2, 1 day samples; lane 3, 3 day samples; lane 4, 7 day samples. (A) GG desmin; (B) DG desmin; (C) GG a-actinin; (D) DG a-actinin. D = Desmin; aA = a-Actinin.
60 40 20 0 0
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Postmortem time, day Fig. 5. Postmortem changes in l/m-calpain activity of goose (GG) and duck (DG) gizzard smooth muscle samples stored at 5 °C. Activity is expressed as percentage of the 0 day GG l/m-calpain activity, which was taken as 100%. Vertical bars show the standard deviation of the means. j: GG; h: DG.
3.3. Postmortem changes in desmin and a-actinin Postmortem degradation of gizzard smooth muscle desmin and
a-actinin (Fig. 6) was examined by Western blotting. The extent of proteolysis of the different proteins was quantified as a percentage of the relative content of the intact protein band at death. The results showed that degradation of desmin was apparently more rapid in GG (Fig. 6A) than DG (Fig. 6B) samples during postmortem storage at 5 °C. As time postmortem proceeded, the relative content of GG desmin was quickly (p < 0.05) reduced in 1 day samples to 58% of that in the 0 day GG samples, to 31% in 3 day samples and to 8% in 7 day samples, respectively. In DG samples, however, the decrease in desmin was (p > 0.05) very slow in 1 and 3 day samples, which remained at 91% and 87% of the desmin present in the 0 day DG samples. An additional decrease (p < 0.05) to 65% in desmin content was seen in 7 day samples. On the other hand, a-actinin was relatively resistant to postmortem proteolysis in both GG (Fig. 6C) and DG (Fig. 6D) samples. The a-actinin content in both samples decreased (p > 0.05) by less than 10% throughout the 7 day storage period.
3.4. General discussion Desmin, a calpain-sensitive protein, is very abundant in smooth muscle. In good agreement with previous studies of postmortem duck skeletal muscle (Chang & Chou, 2012), our results showed that the rate and extent of activation and autolysis of l-calpain, rather than l/m-calpain, paralleled desmin degradation in postmortem gizzard smooth muscle. Moreover, our results also showed
that little change in content of smooth muscle a-actinin, a calpainresistant protein, was observed during postmortem storage. Additionally, ultimate pH in postmortem GG and DG smooth muscles (Fig. 1) was approximately 7, which is also close to the optimal pH for calpain activity. Collectively, these results suggested that l-calpain was closely associated with postmortem proteolysis of gizzard smooth muscle and was similar to that observed in skeletal muscle by Geesink et al. (2006). However, it has been proposed that postmortem proteolysis of skeletal muscle could also be due to a synergistic action of a multienzyme system (Ouali et al., 2006). In addition to calpains, other candidate enzyme systems, which might be involved in postmortem proteolysis of skeletal muscle, mainly include lysosomal cathepsins, caspases and proteasomes. Among them, the lysosomal cathepsins, which are active in acidic pH, might not have played a significant role in the postmortem proteolysis of gizzard smooth muscle because of the neutral ultimate pH in the postmortem gizzard. The caspases, which have a substrate specificity similar to calpains, have been reported to play a role in postmortem proteolysis of skeletal muscle (Herrera-Mendez, Becila, Boudjellal, & Ouali, 2006). However, whether the caspases are active in postmortem skeletal muscle has been a subject of controversy. It was reported that caspase activity and changes of the activity could be detected in early postmortem porcine (Kemp, Bardsley, & Parr, 2006) and ovine skeletal muscles (Kemp et al., 2009), suggesting that the caspases might be active during postmortem storage. In bovine skeletal muscle, in contrast, no significant increase in caspase 3 activity was found during postmortem storage, implying that caspase 3 might not be activated (Underwood, Means, & Du, 2008). Moreover, Kemp et al. (2006) first reported that degradation of desmin was observed in purified porcine skeletal myofibrils incubated with caspase 3. However, recent studies (Mohrhauser et al., 2011) showed no changes in desmin content over time in bovine skeletal myofibrils incubated with caspase 3. In addition, it was reported that energy (or ATP) is required for caspase activation (Zamaraeva et al., 2005) during cell apoptosis. Gizzard smooth muscle contains a limited amount of glycogen (Gröschel-Stewart
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& Zuber, 1990), suggesting that ATP, generated from postmortem glycolysis, might be inadequate. Lack of ATP supply might switch the form of cell death from apoptosis to necrosis, which is believed to be a calpain-mediated process (Syntichaki & Tavernarakis, 2003). Therefore, the involvement of caspases in postmortem proteolysis of gizzard smooth muscle might be very limited, a phenomenon also observed by Kemp et al. (2009), who concluded that the caspases might not play a significant role in postmortem proteolysis of ovine skeletal muscle. On the other hand, the 26S proteasome present in the sarcoplasm of skeletal muscle is a multicatalytic protease complex with optimal activity at pH 7–8 (Foucrier et al., 2001), close to the ultimate pH of postmortem gizzard smooth muscle. In the absence of ATP, the 26S proteasome reversibly dissociates into the 19S domain and the 20S proteasome (Peters, Franke, & Kleinschmidt, 1994). Accordingly, the 20S proteasome, which requires neither ATP nor ubiqutin (Peters et al., 1994), may be potentially generated in postmortem gizzard smooth muscle. A previous study indicated that the proteasome might not have a direct role in the postmortem proteolysis of ovine skeletal myofibrils (Koohmaraie, 1992). Based on the observations of specific structural changes that occurred in high pH calf skeletal muscle, however, a recent study suggested that the 20S proteasome could have a direct role in postmortem proteolysis (Dutaud et al., 2006). Furthermore, the 20S proteasome activity not only degraded skeletal myofibrillar proteins in vitro (Houbak et al., 2008), but also remained very stable at 7 day postmortem in bovine skeletal muscle (Lamare, Taylor, Farout, Briand, & Briand, 2002). These reports suggested that the proteasome might have a role in the postmortem proteolysis of gizzard smooth muscle but more direct evidence would be needed.
4. Conclusion Our results showed that the 0 day pH of approximately 7, in both GG and DG samples, changed little during postmortem storage. Casein zymograms and Western blots showed that activation and autolysis of l-calpain, but not l/m-calpain, paralleled desmin degradation in gizzard smooth muscle during postmortem storage. Moreover, the a-actinin content remained nearly unchanged in postmortem GG and DG samples. Our results, therefore, suggest that l-calpain is actively involved in the postmortem proteolysis of smooth muscle desmin.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
l-Calpain is involved in the postmortem proteolysis of gizzard smooth muscle Ya-Shiou Chang a, Marvin H. Stromer b, Rong-Ghi R. Chou a,⇑ a b
Department of Animal Science, National Chiayi University, Chiayi City, Taiwan Department of Animal Science, Iowa State University, Ames, IA, USA
a r t i c l e
i n f o
Article history: Received 14 August 2012 Received in revised form 9 January 2013 Accepted 28 January 2013 Available online 9 February 2013 Keywords: Gizzard smooth muscle Postmortem proteolysis Calpain Desmin
a b s t r a c t Postmortem changes in proteins that have been implicated in affecting muscle integrity were examined in goose (GG) and duck (DG) gizzard smooth muscle stored at 5 °C. GG and DG smooth muscles were sampled at 0, 1, 3 and 7 day of storage. The pH was approximately 7 in both GG and DG samples during postmortem storage. Casein zymograms showed that 0-day l-calpain activity was higher (p < 0.05) in GG than in DG samples. As postmortem time progressed, l-calpain was activated and autolyzed more extensively in GG than in DG samples. However, l/m-calpain remained relatively stable in both samples. Western blots indicated that postmortem desmin degradation was more rapid in GG than in DG samples. In contrast, a-actinin remained nearly unchanged in both samples. Therefore, our results suggest that l-calpain has an important role in the postmortem proteolysis of gizzard smooth muscle. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Postmortem changes in skeletal muscle stored at 0–5 °C have been extensively studied. The most notable of the changes is the degradation of myofibrillar/cytoskeletal proteins. Ouali et al. (2006) proposed that this proteolysis might involve a multi-enzymatic process. Among the endogenous protease systems in skeletal muscle, evidence has shown that the calpains are able to access their myofibrillar/cytoskeletal substrates and to reproduce the proteolysis pattern observed during postmortem storage of skeletal muscle (Koohmaraie & Geesink, 2006). By fulfilling those criteria, the calpains, especially l-calpain, appear to play an essential role in the postmortem proteolysis of skeletal muscle (Geesink, Kunchay, Chishti, & Koohmaraie, 2006). However, the possible role of other endogenous proteases such as the lysosomal cathepsins (Uytterhaegen, Claeys, & Demeyer, 1994), the proteasome (Houbak, Ertbjerg, & Therkildsen, 2008) and caspases (Kemp, King, Shackelford, Wheeler, & Koohmaraie, 2009; Mohrhauser, Underwood, & Weaver, 2011) have also been implicated. Poultry gizzard is not only a by-product in the poultry industry, but is also a popular food item in oriental cuisine and traditional meat snacks in Asia. The gizzard is mainly composed of smooth muscle and connective tissue (Ruantrakool & Chen, 1986). It was reported that connective tissue (or collagen) did not undergo significant changes during postmortem storage at 0–5 °C (McCormick, 1994). However, little information is available regarding postmor-
tem proteolysis in gizzard smooth muscle stored at 0–5 °C. The purpose of this study, therefore, was to examine whether the calpains were involved in the postmortem proteolysis of goose and duck gizzard smooth muscles stored at 5 °C, and also to compare the differences in postmortem proteolysis between goose and duck gizzard smooth muscles. 2. Materials and methods 2.1. Sample preparation The Institutional Animal Care and Use Committee, National Chiayi University, approved the use of the animals in this study. Gizzards from six White Roman geese and six Pekin ducks were obtained 20 min postmortem from a local slaughter house. Each gizzard was used as one replication. The weight of individual goose and duck gizzards were 140–155 and 115–130 g, respectively. After trimming off any visible connective tissue, each gizzard was immediately cut into four equal portions and allocated to four sampling times. The portions were then vacuum-packed individually and stored at 5 °C for 0, 1, 3, and 7 day. At the end of each storage period, the samples were finely cut with a scalpel and stored in liquid nitrogen until required for subsequent analysis. Duplicate samples from each gizzard, at each storage time, were used for each analysis. 2.2. pH measurement
⇑ Corresponding author. Address: Department of Animal Science, National Chiayi University, 300 University Road, Chiayi City 60083, Taiwan. Tel.: +886 5 2717533; fax: +886 5 2753459. E-mail address: [email protected] (Rong-Ghi R. Chou). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.01.075
A 5 g aliquot from each gizzard sample was crushed in liquid nitrogen for the pH determination by the method of Farouk and Swan (1997). The pH for each sample was measured by a Suntex
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2.3. Casein zymography The procedure used for protein extraction was based on the method of Veiseth, Shackelford, Wheeler, and Koohmaraie (2001). Briefly, a 5 g sample was homogenized in three volumes of extraction buffer (100 mM Tris base, 10 mM EDTA, 0.05% 2mercaptoethanol, pH 8.3) at 5 °C. Homogenates were centrifuged at 22,000 g for 25 min at 5 °C. The protein concentration of the supernatant was determined by using a modified biuret method (Robson, Goll, & Temple, 1968). Zymograms were routinely run in 12% gels (acrylamide:methylenebisacrylamide = 37.5:1, w/w) containing 0.21% casein (w/v) by the method of Raser, Posner, and Wang (1995). The sample buffer (150 mM Tris–HCl, pH 6.8, 20% glycerol, 0.05% 2-mercaptoethanol (MCE), 0.02% [w/v] bromophenol blue) was added to the protein extract at a ratio of 2 parts of the buffer to 3 parts of protein extract (v/v). The same amount of protein (250 lg) from each sample was loaded onto each well of the casein gels. The casein minigels (0.75 mm, Bio-Rad Laboratories, Herculues, CA, USA) were prerun at 100 V for 15 min, 5 °C, with a running buffer containing 25 mM Tris–HCl, 0.05% MCE, 192 mM glycine, and 1 mM EDTA (pH 8.3) before samples were loaded onto the wells. The gels were run at 100 V for 2 h, 5 °C, and then incubated at room temperature in three changes of a 50 mM Tris–HCl (pH 7.5) buffer containing 0.05% MCE and CaCl2 (0.01, 0.03, 0.1 or 4 mM) with slow shaking for 1 h. This was followed by a 16 h incubation in the same buffer at 37 °C. The gels were then stained for 2 h with Coomassie blue (R-250) and destained with 20% methanol and 7% acetic acid. 2.4. Western blot analysis The muscle samples for SDS–PAGE were prepared by the method of Fritz and Greaser (1991). Briefly, muscle samples (0.2 g) were crushed in liquid nitrogen and vigorously mixed with the SDS– PAGE sample buffer (4 ml) containing 8 M urea, 2 M thiourea, 3% SDS, 75 mM dithiothreitol, 0.05% bromophenol blue, and 25 mM Tris–HCl, pH 6.8. The mixture was heated at 100 °C for 3 min and sonicated for 10 min at room temperature. The SDS–PAGE was done in a 10% tris–glycine slab gel (acrylamide:methylenebisacrylamide = 37.5:1, w/w). Equal volume of each sample (20 ll) were loaded onto each well. Proteins were transferred from the 10% slab gel to a nitrocellulose membrane. After the transfer, the membrane was incubated in a 5% bovine serum albumin–phosphate buffer solution (BSA–PBS) for 30 min at 37 °C and was then washed, three times (5 min each), in a 0.1% BSA–PBS solution at room temperature. A monoclonal antibody to desmin (Clone DE-U-10, 1:500 dilution) or a-actinin (Clone BM-75.2, 1:300 dilution) was used as a primary antibody. The membrane was incubated with the primary antibody for 2 h at 25 °C, washed three times (5 min each) in 0.1% BSA–PBS, incubated with a secondary antibody, goat anti-mouseHRP for 2 h at room temperature, washed twice (5 min each) in 0.1% BSA–PBS solution and twice (1 min each) in deionized water. The colour was developed by using SIGMAFASTTM 3,30 -diaminobenzidine tablets. The primary and secondary antibodies, as well as the SIGMAFASTTM 3,30 -diaminobenzidine tablets, were purchased from the Sigma–Aldrich Company (St. Louis, MO, USA).
J131B, Epson), using Photoshop software. Each blot or gel included a pooled 0 day GG sample as a reference standard to normalize the band intensities. The resulting signals were quantified using Image Gauge (version 3.46, Science Lab 99 for Windows, Fuji Film, Tokyo, Japan). The relative l-calpain activity, as well as the relative l/mcalpain activity of 1, 3, and 7 day postmortem GG samples and of 0, 1, 3, and 7 day postmortem DG samples was expressed as a percentage of the relative l-calpain activity and l/m-calpain activity present in the 0 day GG samples. On the other hand, the relative desmin and a-actinin contents in the 0 day GG and DG samples were taken as 100% with respect to each gizzard sample. 2.6. Statistical analysis Split-plot design was used in this study. Whole units were the gizzards from each avian species, and subunits were the smooth muscle samples taken at each sampling time. All data were analyzed using the Mixed Model procedure of SAS (PROC Mixed) (SAS Institute Inc., 2004). The model included avian species, time postmortem and their interaction (species time postmortem) for fixed effects and birds for random effects. A Tukey’s honestly significant difference test was used to compare multiple means. 3. Results and discussion 3.1. Postmortem changes in smooth muscle pH The changes in postmortem pH of goose (GG) and duck (DG) gizzard smooth muscles are shown in Fig. 1. The results showed that the pH was insignificantly different (p > 0.05) between GG and DG samples during a 7 day postmortem storage at 5 °C. The average 0 day pH was 7.08 ± 0.03 in GG samples and was 7.10 ± 0.11 in DG samples and remained nearly unchanged (p > 0.05) in both samples throughout the 7 day sampling period. These results indicated that the ultimate pH in both gizzard smooth muscles was close to 7, which was higher than goose cardiac (6.12) and breast (5.96) muscles (Ho, Lin, & Chou, 2008). It was reported that the glycogen content of gizzard smooth muscle was approximately nine times and two and half times lower than that of skeletal muscle and of cardiac muscle, respectively (Gröschel-Stewart & Zuber, 1990). This suggested that lactic acid production from postmortem glycolysis was limited in gizzard smooth muscle, resulting in a higher ultimate pH. 3.2. Analysis of casein zymography 3.2.1. Activity of l-l/m-, and m-calpain at 0 day Casein zymograms of 0 day GG and DG samples (Fig. 2) showed that one band, with slow migration, clearly appeared in the presence of 10 lM Ca2+ (Fig. 2A). When Ca2+ concentration was 7.5
pH
470 pH meter (Suntex Instruments Co., Taiwan) with a glass electrode.
7.0
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2.5. Image analyses
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Postmortem time, day Either two or three representative casein gels or blots from each gizzard were used for image analysis. The bands in blots or casein gels, which were incubated in the presence of 4 mM Ca2+ to activate all calpain isoforms, were digitized with a scanner (Model
Fig. 1. Postmortem changes in pH of goose (GG) and duck (DG) gizzard smooth muscles stored at 5 °C. Each point (mean ± SD) is the average of six separate replications (n = 6). Vertical bars show the standard deviation of the means. j: GG; h: DG.
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Fig. 2. Zymograms showing l-, l/m-, and m-calpains in the presence of 10 lM (A), 30 lM (B), 100 lM (C), 4 mM (D) calcium, and image analysis results of l- and l/mcalpain activities in the presence of 4 mM calcium in goose (GG) and duck (DG) gizzard smooth muscle 0 day samples (E). Lane 1, GG samples; lane 2, DG samples. l = lcalpain; l/m = l/m-calpain; m = m-calpain. The 0 day GG samples were taken as 100%. Vertical bars show the standard deviation of the means. Different letters within each calpain isoform indicate significant differences (p < 0.05). j: GG; h: DG.
increased to 30 lM, one additional band with an electrophoretic mobility greater than that of l-calpain began to appear (Fig. 2B). These results suggested that the calpain sensitive to 10 lM Ca2+ was l-calpain, and that the calpain with a greater mobility was the l/m-calpain defined by (Sorimachi et al., 1995). Furthermore, no extra bands were found in GG and DG samples in the presence of 100 lM Ca2+ (Fig. 2C). However, an additional band of activity between the l-calpain and the l/m-calpain bands was present in GG, but not in DG, samples in the presence of 4 mM Ca2+ (Fig. 2D). A third calpain, with half-maximal activity at 3.8 mM Ca2+, was purified from mature egg-laying hen skeletal muscle by Wolfe et al. (1989) and might be the skeletal muscle analogue of the additional band we observed in smooth muscle. Sorimachi et al. (1995) proposed that this third calpain might be m-calpain. Therefore, our results imply that, in the presence of 4 mM Ca2+, the additional calpain activity seen in the GG samples might be due to a putative m-calpain. However, this putative m-calpain was absent in the DG samples (Fig. 2D) and in 100 day-old chicken skeletal muscle (Chang & Chou, 2010). Collectively, these results suggested that the presence of this putative m-calpain might depend on avian species and/or animal age. Image analysis of the 0 day zymograms incubated with 4 mM Ca2+ showed that the 0 day activity of l-calpain was 62% higher (p < 0.05) in GG than in DG samples (Fig. 2E). However, no significant difference (p > 0.05) in the 0 day activity of l/m-calpain was found between GG and DG samples (Fig. 2E).
3.2.2. Postmortem changes in l- and l/m-calpain activities Postmortem changes in l- and l/m-calpain activities in GG and DG samples are shown in Fig. 3. The results indicated that l-calpain activity, rather than l/m-calpain activity, appeared to decrease more rapidly in GG than in DG samples as postmortem time progressed (Fig. 3). When the zymograms in Fig. 3 were subjected to image analysis (Fig. 4), the GG l-calpain activity in 1 day
Fig. 3. Zymograms showing postmortem changes in l- and l/m-calpain activities in the presence of 4 mM calcium in goose (A) and duck (B) gizzard smooth muscle samples stored at 5 °C. Lane 1, 0 day samples; lane 2, 1 day samples; lane 3, 3 day samples; lane 4, 7 day samples. l- = l-calpain; l/m- = l/m-calpain; m- = mcalpain.
samples decreased (p < 0.05) to 82% of the 0 day GG l-calpain activity. The level of l-calpain activity continued to decrease (p < 0.05) to 29% and 20% in 3 and 7 day samples, respectively. In contrast, the decrease in l-calpain activity in DG samples was fairly slow (Fig. 4). The DG l-calpain activities were not different (p > 0.05) among the sampling times which were 35% of the 0 day GG l-calpain activity at 0 day, 30% at 1 day, and 27% at 3 day (Fig. 4). The level of l-calpain activity in 7 day DG samples (18% of the 0-day GG l-calpain activity) was below (p < 0.05) from that in 0 day DG samples, but was not different (p > 0.05) from that in 1 and 3 day DG samples. These results indicated that l-calpain activation and autolysis were more rapid and extensive in GG samples than in DG samples. On the other hand, l/m-calpain activity in GG and DG samples remained nearly unchanged (p > 0.05) during the entire 7 day postmortem storage period (Fig. 5).
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Relative activity, %
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Fig. 4. Postmortem changes in l-calpain activity of goose (GG) and duck (DG) gizzard smooth muscle samples stored at 5 °C. Activity is expressed as percentage of the 0 day GG l-calpain activity, which was taken as 100%. Vertical bars show the standard deviation of the means. j: GG; h: DG.
Relative activity, %
120 100 80 Fig. 6. Western blot showing changes in desmin and a-actinin of goose (GG) and duck (DG) gizzard smooth muscle samples during postmortem storage at 5 °C. Lane 1, 0 day samples; lane 2, 1 day samples; lane 3, 3 day samples; lane 4, 7 day samples. (A) GG desmin; (B) DG desmin; (C) GG a-actinin; (D) DG a-actinin. D = Desmin; aA = a-Actinin.
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Postmortem time, day Fig. 5. Postmortem changes in l/m-calpain activity of goose (GG) and duck (DG) gizzard smooth muscle samples stored at 5 °C. Activity is expressed as percentage of the 0 day GG l/m-calpain activity, which was taken as 100%. Vertical bars show the standard deviation of the means. j: GG; h: DG.
3.3. Postmortem changes in desmin and a-actinin Postmortem degradation of gizzard smooth muscle desmin and
a-actinin (Fig. 6) was examined by Western blotting. The extent of proteolysis of the different proteins was quantified as a percentage of the relative content of the intact protein band at death. The results showed that degradation of desmin was apparently more rapid in GG (Fig. 6A) than DG (Fig. 6B) samples during postmortem storage at 5 °C. As time postmortem proceeded, the relative content of GG desmin was quickly (p < 0.05) reduced in 1 day samples to 58% of that in the 0 day GG samples, to 31% in 3 day samples and to 8% in 7 day samples, respectively. In DG samples, however, the decrease in desmin was (p > 0.05) very slow in 1 and 3 day samples, which remained at 91% and 87% of the desmin present in the 0 day DG samples. An additional decrease (p < 0.05) to 65% in desmin content was seen in 7 day samples. On the other hand, a-actinin was relatively resistant to postmortem proteolysis in both GG (Fig. 6C) and DG (Fig. 6D) samples. The a-actinin content in both samples decreased (p > 0.05) by less than 10% throughout the 7 day storage period.
3.4. General discussion Desmin, a calpain-sensitive protein, is very abundant in smooth muscle. In good agreement with previous studies of postmortem duck skeletal muscle (Chang & Chou, 2012), our results showed that the rate and extent of activation and autolysis of l-calpain, rather than l/m-calpain, paralleled desmin degradation in postmortem gizzard smooth muscle. Moreover, our results also showed
that little change in content of smooth muscle a-actinin, a calpainresistant protein, was observed during postmortem storage. Additionally, ultimate pH in postmortem GG and DG smooth muscles (Fig. 1) was approximately 7, which is also close to the optimal pH for calpain activity. Collectively, these results suggested that l-calpain was closely associated with postmortem proteolysis of gizzard smooth muscle and was similar to that observed in skeletal muscle by Geesink et al. (2006). However, it has been proposed that postmortem proteolysis of skeletal muscle could also be due to a synergistic action of a multienzyme system (Ouali et al., 2006). In addition to calpains, other candidate enzyme systems, which might be involved in postmortem proteolysis of skeletal muscle, mainly include lysosomal cathepsins, caspases and proteasomes. Among them, the lysosomal cathepsins, which are active in acidic pH, might not have played a significant role in the postmortem proteolysis of gizzard smooth muscle because of the neutral ultimate pH in the postmortem gizzard. The caspases, which have a substrate specificity similar to calpains, have been reported to play a role in postmortem proteolysis of skeletal muscle (Herrera-Mendez, Becila, Boudjellal, & Ouali, 2006). However, whether the caspases are active in postmortem skeletal muscle has been a subject of controversy. It was reported that caspase activity and changes of the activity could be detected in early postmortem porcine (Kemp, Bardsley, & Parr, 2006) and ovine skeletal muscles (Kemp et al., 2009), suggesting that the caspases might be active during postmortem storage. In bovine skeletal muscle, in contrast, no significant increase in caspase 3 activity was found during postmortem storage, implying that caspase 3 might not be activated (Underwood, Means, & Du, 2008). Moreover, Kemp et al. (2006) first reported that degradation of desmin was observed in purified porcine skeletal myofibrils incubated with caspase 3. However, recent studies (Mohrhauser et al., 2011) showed no changes in desmin content over time in bovine skeletal myofibrils incubated with caspase 3. In addition, it was reported that energy (or ATP) is required for caspase activation (Zamaraeva et al., 2005) during cell apoptosis. Gizzard smooth muscle contains a limited amount of glycogen (Gröschel-Stewart
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& Zuber, 1990), suggesting that ATP, generated from postmortem glycolysis, might be inadequate. Lack of ATP supply might switch the form of cell death from apoptosis to necrosis, which is believed to be a calpain-mediated process (Syntichaki & Tavernarakis, 2003). Therefore, the involvement of caspases in postmortem proteolysis of gizzard smooth muscle might be very limited, a phenomenon also observed by Kemp et al. (2009), who concluded that the caspases might not play a significant role in postmortem proteolysis of ovine skeletal muscle. On the other hand, the 26S proteasome present in the sarcoplasm of skeletal muscle is a multicatalytic protease complex with optimal activity at pH 7–8 (Foucrier et al., 2001), close to the ultimate pH of postmortem gizzard smooth muscle. In the absence of ATP, the 26S proteasome reversibly dissociates into the 19S domain and the 20S proteasome (Peters, Franke, & Kleinschmidt, 1994). Accordingly, the 20S proteasome, which requires neither ATP nor ubiqutin (Peters et al., 1994), may be potentially generated in postmortem gizzard smooth muscle. A previous study indicated that the proteasome might not have a direct role in the postmortem proteolysis of ovine skeletal myofibrils (Koohmaraie, 1992). Based on the observations of specific structural changes that occurred in high pH calf skeletal muscle, however, a recent study suggested that the 20S proteasome could have a direct role in postmortem proteolysis (Dutaud et al., 2006). Furthermore, the 20S proteasome activity not only degraded skeletal myofibrillar proteins in vitro (Houbak et al., 2008), but also remained very stable at 7 day postmortem in bovine skeletal muscle (Lamare, Taylor, Farout, Briand, & Briand, 2002). These reports suggested that the proteasome might have a role in the postmortem proteolysis of gizzard smooth muscle but more direct evidence would be needed.
4. Conclusion Our results showed that the 0 day pH of approximately 7, in both GG and DG samples, changed little during postmortem storage. Casein zymograms and Western blots showed that activation and autolysis of l-calpain, but not l/m-calpain, paralleled desmin degradation in gizzard smooth muscle during postmortem storage. Moreover, the a-actinin content remained nearly unchanged in postmortem GG and DG samples. Our results, therefore, suggest that l-calpain is actively involved in the postmortem proteolysis of smooth muscle desmin.
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