Results and discussion

Gels were run at 20 V overnight, then stained using ProQ Diamond Phosphoprotein Stain

(Molecular Probes Inc., Eugene, OR), following the manufacturer’s protocol. Following imaging for phosphoproteins gels were stained with SyPro Ruby Total Protein Stain (Molecular Probes Inc., Eugene, OR) following the manufacturer’s protocol. Gels were then imaged using a ChemiImager 5500 (Alpha Innotech, San Leandro, CA).

Immunoblotting of 2D gels was done to characterize calpastatin peaks from Semitendinosus muscle using 80 µg of protein per strip and were, resolved using 7 cm immobilized pH gradient strip (pH 4 to 7; GE Healthcare) following protocol for rehydration as previously described. The isoelectric focusing was performed on an Ettan IPGphor isoelectric focusing system (GE Healthcare) for a total of 7,000 V h. Strips were loaded onto a 12.5 % SDS-PAGE gels, transferred to PVDF membrane and imaged following the previously described protocol. The primary antibody anti calpastatin (MA3-944, Thermo Scientific, Rockford, IL) was used in the dilution 1:5,000, and a goat anti-mouse horseradish peroxidase (No 2554, Sigma, St. Louis, MO) was used of a secondary antibody at 1:10,000 dilution.

2.3. Statistical analysis

Data from calpastatin peaks, calpain-1 and calpain-2 activity and abundance of bands from Western blot were analysed using split plot design in a repeated measures arrangement. Animal as the whole plot, muscle as the split plot and days post mortem were used as repeated measures. Statistical analysis was performed using statistical software R (R Development Core Team, Vienna, AU). Gel images from 2D-DIGE experiments were analyzed using Decider (version 6.5, GE Healthcare, Piscataway, NJ). A P-value < 0.10 was considered statistically significant. Significant spots present in more than 83% of the images were initially selected for identification.

3.1. Experiment 1

3.1.1. Activity of CAST peak 1 and 2, calpain-1 and calpain-2.

In all extractions, the elution pattern of both peaks of calpastatin, calpain-1 and calpain-2 was similar (Figure 1). The first peak of calpastatin was eluted in a range of 60 to 90 mM KCl followed by second peak of calpastatin eluted in a range of 120 to 180 mM KCl, calpain-1 eluted in a range of 185 to 260 mM KCl and calpain-2 eluted in a range of 330 to 400 mM KCl. This pattern of elution that shows two peaks of calpastatin has been reported before (Pontremoli et al., 1992;

Salamino et al., 1994; Geesink, Nonneman and Koohmaraie, 1998; M Averna et al., 2001; Cruzen et al., 2013). However, the exact characteristics of the calpain inhibitory species of those peaks are not known.

Possible reasons for these two peaks include post translational modifications (potentially

phosphorylation) of calpastatin (Pontremoli et al., 1992; M Averna et al., 2001) that could change the ionic charge of the molecule and modify its affinity for the column and thus its elution time.

Another possibility could be an alternative splicing of the gene (Geesink, Nonneman and

Koohmaraie, 1998; Gaarder, Thomassen and Veiseth-Kent, 2011b). Degradation of the calpastatin molecule is another possibility for the separation of two peaks and could be an explaination for some reports of lost activity during the purification process (Geesink, Nonneman and Koohmaraie, 1998).

Aging time post mortem resulted in an increase in CAST 1 from the Triceps brachii (TB) (P<0.05), but had no significant influence on the CAST 1 from Longissimus lumborum (LL) (Table 1). On the other hand, total CAST and CAST 2 activity decreased during post mortem aging in both

muscles. The drop in total CAST activity was mostly accounted for the CAST 2 decrease in activity during aging. This could indicate that CAST 2 is the predominant form of CAST that is measured during traditional assays.. This activity loss has been attributed to CAST degradation by calpain-1 and/or calpain-2 or other proteases (Doumit and Koohmaraie, 1999; De Tullio et al., 2000). The

hypothesis that post mortem CAST 2 degradation would result in CAST 1 was not corroborated by activity level, unless it could be considered that CAST 1 inhibitory efficiency would be lower than CAST 2 for calpain-2 (which was used as the positive control in the assays). It could also depend on the phosphorylation status of the two fractions (Pontremoli et al., 1992; Salamino et al., 1994).

Calpain-1 activity was greater in LL than TB at day 0 and decreased more rapidly during post mortem aging in LL (P<0.05). Calpain-2 activity had no change (P>0.05) during post mortem aging or among muscles. This is consistent with previous observations (Koohmaraie et al., 1987). The ratio of CAST 2 and CAST total to calpain-1 activity was greater in TB than in LL (Table 2). That result may provide a partial explanation for the more rapid decline of calpain-1 activity in LL, since greater CAST activity seems to spare calpain-1 from post mortem autolysis and activity loss

(Delgado et al., 2001), enabling the possibility of LL to exhibit more proteolysis and tenderness than TB. Interaction between calpain and calpastatin is the most relevant mechanism involved in the tenderization process during post mortem period mediated by cellular structural proteins (Melloni et al., 2006). A similar difference between LL and TB in calpain-1 / calpastatin ratio was found by (Cruzen et al., 2014).

3.1.2. SDS-PAGE and immunoblotting

In western blots for CAST bands between 115 kDa and 36 kDa were detected (Figure 2). Only the bands presenting strong signal were analyzed. There was no detectable difference between muscles for the 115 kDa band of CAST (Table 3), the size reported for intact calpastatin (Takano et al., 1986; Nakamura et al., 1989; Cruzen et al., 2014). The 115 kDa band of CAST decreased in intensity during post mortem aging in both muscles (P<0.05), with more than 70% of the change occurring in the first day. At this same time, the abundance of 90 kDa band of CAST increased, while there were no differences between day 0 and 7 for both muscles. At day 1, the 90 kDa band of CAST was more abundant in the LL than in the TB. Although the 70 kDa band of CAST also

increased at day 1 for TB, differences in it were not detected for the LL between day 0 and 1.

Moreover, the abundance of the 90 kDa band of CAST was greater in TB than LL. Another difference between muscles was observed for the 45 kDa band of CAST. The 45 kDa band of CAST was more abundant in LL than TB over all days and did not change in abundance in the same muscle over the aging period. Overall, the appearance of the two more intense bands of CAST probably are result of degradation of the intact calpastatin, even though there was some differences between muscles. A similar pattern of bands was found using the products of purified calpastatin incubated with calpain-1 and calpain-2 (Doumit and Koohmaraie, 1999).

The autolysis of the 80 kDa band of calpain-1 in the sarcoplasmic fraction increased in both muscles during post mortem aging. Autolysis decreases the requirement of free Ca²⁺ for calpain activity from 3-50 µM to 0.5-2 µM Ca²⁺ (Goll et al., 2003) even though it also decreases its stability at the higher ionic strengths found in post mortem muscle (Geesink and Koohmaraie, 1999b). Nonetheless, the autolysis is associated with activation of calpain and could provide information about the proteolysis process in the muscle. The appearance of the 78 kDa autolyzed calpain at day 1 was more pronounced in the LL compared to the TB, with a decrease at day 7 of this band compared to day 1. In the TB, a decrease in the 78 kDa autolysis product of the catalytic subunit was not detected between days 1 and 7 postmortem (Figure 3). The proportion of the catalytic subunit present as the 76 kDa autolysis product increased at day 7 for both muscles with greater amount in the LL. These changes in the sarcoplasmic fraction point to a slower rate of autolysis in the TB, which might be associated to the greater calpastatin / calpain-1 ratio in that muscle and perhaps a slower rate of protein degradation.

The calpain-1 is present in soluble in the sarcoplasmic fraction and during meat aging start to be associated to myofibrils at the Z-line and A-band (Melody et al., 2004). The reason for appearance of myofibril-bound calpain-1is not clear, and remains certain proteolytic activity during post mortem aging period (Delgado et al., 2001). In the myofibrillar fraction, the decrease of the 80 kDa band of intact calpain-1 happened after 24 hours post mortem and in day 7 post mortem this

band was not abundant. The 78 kDa band of calpain-1 was increased in both TB and LL at day 1, but it decreased at day 7 in LL; the 78 kDa band of autolyzed calpain-1 product was greater at day 7 compared to day 0 in TB. The abundance of 76 kDa band of autolyzed calpain-1 was increased at day 7 in the myofibrillar fraction for both muscles. Therefore, autolysis of calpain-1 presents the same pattern between sarcoplasmic and myofibrillar fractions. Similar autolysis progression in both sarcoplasmic and myofibrillar fractions has been reported before (Melody et al., 2004; Rowe et al., 2004).

Autolyzed bands of calpain-1across aging appear be more abundant in the myofibrillar fraction than in sarcoplasmic fraction, suggesting that myofibril bound calpain-1 is less prone to continued

autolysis compared to the soluble enzyme present in sarcoplasmic fraction. Proteolysis in LL seems to be more rapid than in the TB because at day 1 the 78 kDa band of calpain-1 is more abundant than day 0 in the LL, which could mean more activity in LL early post mortem.

The 30 kDa band in myofibrillar fraction, a troponin T degradation product, is increased at day 7 compared to day 0 for LL, and was more abundant at day 7 in the TB than LL (P<0.05). In this muscle there was no difference between days 1 and 7 (Figure 4). Those results suggest that

proteolysis occurred at a more rapid rate in the LL. The connection of postmortem tenderization of LL to the appearance of 30 kDa band of troponin-T has been reported (Geesink and Koohmaraie, 1999a; S M Lonergan, Rowe, et al., 2001), and it probably could be related to proteolytic activity of calpain-1. The current results together with the current literature suggests that variation in

calpastatin activity explains a portion of the differences in calpain activity and postmortem protein degradation.

The degradation of intact band of desmin starts after 24 hours of aging in LL and this band is more degraded in LL than TB at day 7 (Table 3). For the TB, no difference in the abundance of intact desmin was detected (P<0.05) across days of aging. However, there was more abundance of bands of degraded products of desmin (38 kDa and 35 kDa) at day 7 (Figure 5).

Similar degradation pattern of desmins and troponin-T degradation have been reported in aged beef and purified myofibrils incubated with calpain-1 (Olson and Parrish, 1977; Huff-lonergan et al., 1996). This degradation of desmin and troponin-T is highly related to beef tenderization (Huff-Lonergan, Parrish and Robson, 1995; Geesink et al., 2006).

3.1.3. Two-Dimensional Difference in Gel Electrophoresis

A two-dimensional difference in gel electrophoresis gel (2D-DIGE) experiment showing spots that were differentially abundant in Longissimus lumborum (LL) and Triceps brachii (TB) muscles in sarcoplasmic fraction is summarized in figure 6. Coverage percentage of identified peptides from intact protein and identification of proteins present in each picked spot is presented in table 4.

An interesting finding in this analysis is the greater amount of 70 kDa heat shock proteins (HSP) in the sarcoplasmic fraction of aged beef. The HSP protein family is described as having an important role in post mortem meat aging (Ouali et al., 2006; Carvalho et al., 2014). This protein family is related to meat toughness and has been suggested to be a toughness marker due their cellular protective function against apoptosis. During meat aging, the HSP 70 protein family acts in an anti apoptotic function in the stress response, acting on the caspase-independent pathway and caspase dependent pathway at both ways, upstream and downstream of caspase activation (Creagh, Carmody and Cotter, 2000; Mayer and Bukau, 2005). A signal like cell damage for example, induces the HSP 70 prevent the oligomerization of an apoptotic protease activating factor-1 (Apaf-1), reventing maturation of caspase-9, this process could help the sarcomere maintenance and organization (Beere, 2004; Ouali et al., 2006; Picard et al., 2010).

HSP 70 is normally located aggregated in cytoplasm in non stressed live tissue and after slaughter attached to actin and α-actinin (Margulis and Welsh, 1991; Tupling et al., 2004). The presence of more HSP 70 in day 7 sarcoplasmic fraction could be related to release of this protein from

myofibrillar fraction during post mortem aging probably due the proteolytic action. Under electrical

stimulation the relative abundance of HSP 70 bound to myofibrils is lower than non electrical stimulated one and 24 hours after the slaughter, suggesting eletrical stimulation influences solubility and fractionation of this protein (Bjarnadóttir et al., 2011).

Structural proteins decreased the relative abundance in day 7 compared to day 0 in both muscles in sarcoplasmic fraction. This effect could occur by protease activity upon proteins like alpha4A chain tubulin, desmin and alpha actin that are soluble in sarcoplasmic fraction and being degraded even in the soluble fraction and the fragments are not detectable by the current used techinique.

Myosin light chain (MLC) could be released to sarcoplasmic fraction because of degradation of proteins associated with MLC during post mortem aging (Anderson, Lonergan and Huff-Lonergan, 2012). In fact, Anderson et al. (2012) also demonstrated that incubation of myofibrils with calpain-1 resulted in release of MLC from the myofibris. This could be related to the weakening of the actomyosin and may be an important contributor to tenderization during aging time. The appearance of MLC in the soluble fraction was related to the tenderization process and was negatively related to tender meat 72 hours after slaughter but positively related at 14 days post mortem (Zapata, Zerby and Wick, 2009).

The mitochondrial protein ATP synthase (subunit beta) was increased and Succinyl-CoA ligase decreased in both muscles at day 7 compared to day 0. Adenylate kinase isoenzyme also decreased in the LL at day 7 compared to day 0.The conversion of ADP to ATP is mediated to ATP synthase present in the mitochondria and was found to be more abundant in aged pork meat (Bernevic et al., 2011). Mitochondrial proteins found in the soluble fraction are related to the beginning of the apoptotic process in early post mortem stages and those proteins may be related to the tenderization process (Laville et al., 2009). Apoptosis has been suggested to be an early event related to the tenderization process after slaughter, starting the cell protection machinery (Longo et al., 2015) and ultimately affecting the tenderization process. Another interesting protein that was increased at day 7 in both muscles compared to day 0 is Prostaglandin reductase 2 (Table 4). This enzyme catalyzes the NADPH dependent reduction of 15-keto-prostaglandin E2. Recently, 15-keto-prostaglandin E2

was proposed to trigger the translocation of the pro apoptotic protein Bax to mitochondria, thereby inducing apoptosis (Yun-Chia Chang et al., 2012). This finding points to another protein in the apoptotic pathway that changes during proteolysis post mortem.

3.2. Experiment 2

3.2.1. Phosphoprotein and Total Protein Staining

In the total protein stain assay, the Semitendinosus muscle (ST) showed the same pattern of bands as the TB and proteins from pooled fractions that have active calpastatin from the ST ,CAST 1 and CAST 2, are stained (Figure 7 A). CAST 1 had two bands a 115 kDa band and one upper band that was approximately 125 kDa, while CAST 2 had only an approximately 125 kDa band. Both bands are poorly phosphorylated (Figure 7 B). Although the CAST 1 pool had more total protein than CAST 2 pool, they had a similar pattern of bands, with also the same phosphorylation pattern.

Obviously, CAST1 showed more phosphorylated bands which may be explained by greater amount of total protein. Those differences are due to application of the same amount of eluted volume from each pooled fractions from each calpastatin peak. This approach was taken because CAST 1 is extremely unstable, and it was decided to minimize the manipulation of the samples, with methods such as those for protein concentration.

Calpastatin immunoblots show CAST 2 had a band that migrated with an apparent molecular weight greater than 115kDa and CAST 1 showed a 115kDa band (Figure 8). CAST 2 had a more abundant 70 kDa band than CAST 1. This seems to be important considering the observation that band was present in greater amount in TB at day 1 in experiment 1. This could corroborate that calpastatin variants present in CAST 2 are more relevant to slowing down post mortem degradation.

In Semitendinous muscle from cattle, domain IV, 1xb exon and XL domain was found in a band that was approximately 70 kDa and was attributed to a cleavage in the inhibitory domain II of the

calpastatin molecule. This cleavage generates two degradation products that migrate to similar location in a polyacrylamide gel (Raynaud et al., 2005). Therefore the cleavage could form two distinct products with different affinity for an anion exchange column thus generating the two peaks.

The western blot results from Semitendinosus to detect CAST peaks fractions (Figure 8) were not consistent with the explanation of the conversion of CAST 2 to CAST 1 occurring since some fragments of the CAST 1 fraction may be even larger than what was found in CAST 2. Nonetheless, a partial conversion should not be dismissed.

The two dimensional western blot of calpastatin peaks are presented in figure 9. The sample for the first peak, CAST 1, showed more spots at different isoelectric points and molecular weight than CAST 2 in a 2D immunoblot for calpastatin. Those blots make it difficult to consider that CAST 1 would be only a product of degradation of CAST 2, since there are high molecular weight spots at acidic isoeletric regions in CAST 1 immunoblots. The reason for not identifying high molecular weight spots in CAST 2 needs to be studied. Nonetheless, it could be more plausible to understand those peaks as result of different transcripts variants associated with post-translation modifications, which both may change the CAST proteolytic products that compose those peaks.