Food Chemistry 372 (2022) 131347

Available online 7 October 20210308-8146/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Sarcoplasmic and myofibril-bound calpains during storage of pork longissimus muscle: New insights on protein degradation

Jian Lyu , Per Ertbjerg *

Department of Food and Nutrition, University of Helsinki, 00014 Helsinki, Finland

A R T I C L E I N F O

Keywords: Myofibril-bound calpain Postmortem proteolysis Calpain-1 Calpain-2 Desmin Calcium

A B S T R A C T

The subcellular distribution of calpain-1 and -2 and the proteolytical activity of myofibril-bound calpains in pork were investigated during 12 days cold storage. The content of sarcoplasmic calpain-1 decreased during storage while myofibril-bound calpain-1 content first increased (P < 0.05) to 17% of that of 12 h-sarcoplasmic calpain-1 on day 6 followed by a gradual decrease with subsequent storage, suggesting that calpain-1 gradually trans-located from sarcoplasm to myofibrils during the initial 6 days of postmortem storage. Intact desmin decreased (P < 0.05) after incubation of myofibrils with 0.05 mM Ca2+, and this was more pronounced with 5 mM Ca2+ (P < 0.05). Ca2+ titration curves of day 6 myofibrils showed two distinct proteolytic activities becoming activated in the range 0.03 to 0.06 mM and 0.4 to 0.8 mM Ca2+, respectively. The results suggest that both calpain-1 and calpain-2 binds to myofibrils during storage and subsequently degrade structural proteins including desmin.

1. Introduction

Meat tenderness is one of the most important eating quality char-acteristics (Shackelford, Wheeler, Meade, Reagan, Byrnes, & Koohmar-aie, 2001). Postmortem degradation of key cytoskeletal proteins is a crucial event affecting meat tenderization (Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). The roles of the calpain system in degra-dation of cytoskeletal proteins and in meat tenderization are well recognized (Bhat, Morton, Mason, & Bekhit, 2018; Huff-Lonergan & Lonergan, 2005). Calpain-1 and − 2 and their inhibitor calpastatin, are the most studied in the calpain system. Both calpains are Ca2+ depen-dent and cleave the same substrates. However, the requirement of calpain-1 and calpain-2 on Ca2+ concentration differs, being 3–50 μM and 400–800 μM for half-maximal activity, respectively (Goll, Thomp-son, Li, Wei, & Cong, 2003).

Desmin is an important structural protein and is very susceptible to proteolytic degradation by the calpain system (Nelson & Traub, 1982). As an important intermediate filament protein, desmin builds up a network to connect myofibrils at the Z-disk level and plays a role in organizing the structure of myofibrils within the muscle fiber (Capeta-naki, Milner, & Weitzer, 1997). Thus, desmin degradation is believed to contribute to meat tenderization due to its role in maintaining the structural integrity of the muscle fiber (Huff-Lonergan, Mitsuhashi, Beekman, Parrish, Olson, & Robson, 1996; Pearce, Rosenvold,

Andersen, & Hopkins, 2011). The extractable activity of calpain-1 as assayed in vitro has been

shown to decrease rapidly early postmortem in beef (Koohmaraie et al., 1987; Camou, Marchello, Thompson, Mares, & Goll, 2007). In pork, calpain-1 is partly autolyzed in less than one day (Pomponio & Ertbjerg, 2012; Zhang & Ertbjerg, 2018). The decreased extractable activity in vitro and the emergence of autolyzed forms of calpain are thought to be indicative of proteolytic activity (Goll et al., 2003; Melody, Lonergan, Rowe, Huiatt, Mayes, & Huff-Lonergan, 2004). However, only little degradation of myofibrillar proteins like desmin (Kristensen & Purslow, 2001), troponin-T (Huff-Lonergan et al., 1996) and titin (Fritz & Greaser, 1991) occurs during the first 24 to 48 h after death. Further-more, the extractable activity of calpain-1 decreases more rapidly than that of calpastatin (Boehm, Kendall, Thompson, & Goll, 1998), which means that even if the Ca2+ concentration in the sarcoplasm is enough to activate calpain-1, its activity may still be inhibited by calpastatin to a fairly large extent. The activation of calpain-2 can be increased by high muscle temperature early postmortem (Liu, Ruusunen, Puolanne, & Ertbjerg, 2014; Pomponio & Ertbjerg, 2012), by extended aging time (Colle & Doumit, 2017) and by the freezing-thawing process (Zhang & Ertbjerg, 2018), but calpain-2 is activated later than calpain-1 due to the insufficient Ca2+ concentration in sarcoplasm. Collectively, it is difficult to explain the time difference between decrease of calpain-1 activity and degradation of cytoskeletal proteins without consideration of other

* Corresponding author. E-mail address: per.ertbjerg@helsinki.fi (P. Ertbjerg).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2021.131347 Received 4 April 2021; Received in revised form 2 October 2021; Accepted 4 October 2021

Food Chemistry 372 (2022) 131347

2

enzymes. Detailed knowledge on the process of calpain-induced pro-teolytic degradation of structural proteins and the subsequent contri-bution to meat tenderness is lacking. Therefore, in order to add to the understanding of the mechanisms responsible for meat tenderization during storage, we investigated the distribution of calpains within the muscle fiber with respect to being free in the sarcoplasm or bound to myofibrils and the ability of myofibril-bound calpains to degrade desmin and the effect of Ca2+ concentration on the activity of myofibril-bound calpains.

2. Material and methods

2.1. Sampling processing

Five pigs were slaughtered at a commercial slaughterhouse in Finland (HK-Ruokatalo, Forssa) and pork loins from different animals were excised at 6 h post-mortem, vacuum-packaged and transported on ice to meat laboratory at University of Helsinki. The longissimus thoracis (LT) muscles were trimmed of visible connective tissue as well as fat and 400 g from each muscle was cut into small pieces (about 2 cm × 2 cm ×1 cm) and the samples were vacuum-packaged and then stored at 2 ±1 ◦C in a cold room for 1, 3, 6, 9 and 12 days. Each animal was repre-sented at every storage period. After storage, all of the samples were frozen at − 80 ◦C until use.

The average pH of muscles at 24 h post-mortem was 5.49 ± 0.04 measured by an insertion electrode (Mettler-Toleda Inlab 427). L*, a* and b* average values were 53.1 ± 0.5, 9.0 ± 1.5 and 5.3 ± 0.8, respectively, measured by a Minolta Chroma meter CR-400 (Minolta Camera Co. Ltd., Osaka, Japan) set at D65 illuminant, aperture diameter 8 mm and calibrated using a white tile (C: Y = 93.6, x = 0.3130, y =0.3193). The pH and color data thus suggested that none of the muscles were affected by DFD or PSE.

2.2. Preparation of supernatant and myofibrils

To study the relative amount of calpains being free in the sarcoplasm or being bound to myofibrils, samples from each muscle were fraction-ated into a soluble supernatant and a myofibril pellet. Myofibrils were prepared as described by (Liu, Arner, Puolanne, & Ertbjerg, 2016) with slight modifications. Briefly, meat samples were mixed 1: 6 (w : v) with cold extraction buffer (100 mM Tris-HCl, 5 mM EDTA, 10 mM mono-thioglycerol, pH 8.3) and homogenized by an IKA Ultra-Turrax T25 homogenizer (Labortechnik, Staufen, Germany) at 13,500 rpm for 3 ×12 s, 12 s cooling between bursts, and were then centrifuged at 10,000 × g for 30 min at 4 ◦C. Addition of 5 mM EDTA in the extraction buffer aimed at preventing activation of calcium activated proteases and the whole extraction process was performed below 4 ◦C to slow down enzymatic reactions. We did not use anti-proteases cocktail during the preparation and this might have affected the obtained results. The su-pernatant was collected after centrifugation and the pellet was then washed twice with cold incubation buffer (75 mM KCl, 100 mM Tris, 2 mM MgCl2, pH 7.0) by centrifuging at 10,000 × g at 4 ◦C for 5 min. The washed myofibrils were re-suspended in incubation buffer. The protein concentrations of the collected supernatant and the myofibril suspension from each sample were measured by the RC DC Protein Assay Kit (Bio- Rad Laboratories, Hercules, CA). The extracts were placed on ice and added sample buffer as soon as possible after protein determination. To obtain representative calpain isoforms and to compare the difference between animals at the later time points, the supernatant of samples from all five muscles at 12 h post-mortem was mixed and collected and used as a reference standard in western blots. Changes related to pro-cesses that began before 12 h postmortem were thereby not estimated.

2.3. Activity of myofibril-bound proteases

The potential of myofibril-bound calpains to degrade desmin

following activation by Ca2+ was investigated during the aging period by using 0.05 mM Ca2+ to activate calpain-1 and 5 mM Ca2+ to activate calpain-1 and calpain-2 combined. After each storage time, 1.2 g meat from each loin was finely chopped and then mixed 1:4 (w:v) with cold rigor buffer (75 mM KCl, 100 mM Tris-HCl, 2 mM MgCl2, 2 mM EGTA, pH 7.5). The mixture was homogenized at 13,500 rpm for 3 × 12 s, 12 s cooling between bursts, and was then centrifuged at 10,000 × g for 10 min at 4 ◦C. The myofibrils were washed twice with incubation buffer centrifuging at 10,000 × g at 4 ◦C for 5 min.

The myofibrils were re-suspended in incubation buffer at a concen-tration of 15 mg protein/mL and 2 mL myofibril suspension was added CaCl2 to a final Ca2+ concentration of 0, 0.05 and 5 mM. Then the mixture was incubated at room temperature for 2 h and the reaction stopped by adding 1 mL EGTA buffer (20 mM EGTA, 75 mM KCl, 100 mM Tris, 2 mM MgCl2, pH 7.0). All myofibril pellets were kept on ice before being processed for western blot and quantification of the native (intact) desmin.

To obtain further insight into the proteolytic potential of myofibril- bound proteases, day 6 myofibrils were isolated and Ca2+ titration curves obtained by observing a) release of peptides from the myofibrils and b) desmin degradation. For the titration assay, 0.6 mL myofibril suspension in incubation buffer was added CaCl2 to a final concentration of 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.6 mM. After 2 h-incubation at room temperature, the reaction was stopped by adding 0.3 mL EGTA buffer and centrifuged at 20,000 × g for 8 min. The supernatant was collected for protein content determination by measuring absorbance at 280 nm by taking a reading of 1.00 as 1.00 mg/mL. The myofibril pellets were kept on ice until processed for western blot and quantification of intact desmin.

2.4. SDS-PAGE and western blot

SDS-PAGE and western blot were run according to the method described by (Lyu & Ertbjerg, 2021) with slight modification. After electrophoresis, proteins in gels were transferred to 0.45 μm Nitrocel-lulose Membranes (Thermo sciencetific, Bremen, Germany) in XCell IITM

Blot Module with NuPAGE® Transfer Buffer (20X) from Invitrogen. The blotting process was performed overnight at 30 V for calpains. Mem-branes were then blocked for 1 h in 20 mL of TBS (50 mM Tris, 150 mM NaCl, pH 7.5) with 50 g/L skim milk powder at room temperature. The membranes were incubated with primary antibodies for calpain-1 (MA3- 940 Invitrogen, Carlsbad, CA), calpain-2 (ab39165 abcam, Cambridge, UK) and desmin (DE-U-10 Invitrogen, Carlsbad, CA) at a dilution of 1:1000 in TBST (50 mM Tris, 150 mM NaCl, 0.5 g/L Tween-20, pH 7.5) at room temperature for 2 h. Afterwards, the membranes were washed for 3 × 10 min and incubated for 1 h with secondary antibodies, which were 1.0 μL IRDye® 800 CW Donkey anti-mouse, 2 μL IRDye® 800 CW Donkey anti-rabbit and 1.0 μL IRDye® 800 CW Donkey anti-mouse in 15 mL TBST with 20 g/L skim milk powder for calpain-1, calpain-2 and desmin at room temperature, respectively. After the incubation was completed, membranes were washed in TBST for 10 min twice and in TBS for 10 min once.

After washing, membranes were scanned and quantified by Odyssey Infrared Imaging System-CLx (LI-Cor Cop, Lincoln, NE) using the 800 nm channel. Relative intensity of calpains bands (80-, 78- and 76- kDa bands altogether) was calculated by LI-COR Odyssey program. The fluorescence intensity of each band was calculated based on the inte-grated intensity divided by the mean value of the 12 h standard. The background was calculated using the average method. The results for the content of calpains were expressed as relative intensity (%). For the calpains in the sarcoplasm, the reference standard with the same content as 12 h samples was taken as 100%; for the myofibril-bound calpains, only 25% of the reference standard (compared to sarcoplasmic samples) was loaded due to the lower content of calpain-1 in the myofibril frac-tion. The content of myofibril-bound calpain-1 was corrected to be comparable to the muscle content of sarcoplasmic calpain-1 using a

J. Lyu and P. Ertbjerg

Food Chemistry 372 (2022) 131347

3

myofibril:sarcoplasm protein ratio of 2:1.

2.5. Statistical analysis

Muscles of the different animals were defined as random factor. For each muscle, duplicates were performed for the western blot of calpain-1 and calpain-2. Duplicates were conducted in the incubation experiment. Triplicates were done for the protein content assay. Data analysis was carried out using the IBM SPSS Statistics 25 software. Bonferroni test was conducted to evaluate the significant differences between group means at a level at P < 0.05.

3. Results

3.1. Sarcoplasmic and myofibril-bound calpains

Fig. 1 illustrates the effect of storage on the content and location of calpain-1 in pork. Native calpain-1 was observed only at 12 h and day 1 postmortem and there was only autolyzed forms of calpain-1 in the sarcoplasm of samples from day 3 to 12 (Fig. 1A). The content of sarcoplasmic calpain-1 on day 1 was 71% of that at 12 h, and during cold storage it continued to decrease to 33% at the end of the storage. The decrease was faster in the first 6 days postmortem (P < 0.05) than that of the later storage (Fig. 1B). The content of calpain-1 bound to myofibrils initially increased with storage to reach a maximum at day 6, but thereafter declined. The amount of myofibril-bound calpain-1 at day 6 was 17% of 12 h-sarcoplasmic calpain-1, which was significantly (P <0.05) higher than that of the other days (Fig. 1D). The amount of sarcoplasmic calpain-2 showed a parallel but slightly slower decreasing trend compared to that of calpain-1 (Fig. 2) and the content declined to 39% of that at 12 h. The sensitivity of the western blot for calpain-2 was not high enough to demonstrate the development with time of myofibril- bound calpain-2 (data not shown).

3.2. Desmin

The content of intact desmin in the myofibrils significantly decreased (P < 0.05) during refrigerated storage. There was only 16% of intact desmin left after 12 day-storage (Fig. 3). Myofibrils were isolated by the end of each storage period and then incubated with different

concentrations of Ca2+ to show the activity of myofibril-bound proteases having different requirements for Ca2+. The content of intact desmin decreased after incubation of myofibrils with 0.05 mM Ca2+ compared with that of incubation without Ca2+, and further decreased when the Ca2+-concentration was increased by a factor of 100 to 5 mM (Fig. 4). The data demonstrate that myofibril-bound Ca2+-activated proteases with both high and low sensitivity to Ca2+ were present throughout the storage period in sufficient quantity to induce desmin degradation upon Ca2+ addition.

ab

b

c

ab

a

0

5

10

15

20

25

1 3 6 9 12

Myo

fibril

-bou

nd c

alpa

in-1

(%)

Storage time (days)

(D) (B)

(A) (C)

a

b

cd de

e

0

30

60

90

120

0.5 1 3 6 9 12

niaplacci

msalpocraS-1

(%)

Storage time (days)

Fig 1. Representative western blot of calpain-1 from sarcoplasmic (A) and myofibrillar (C) protein fraction at 0.5, 1, 3, 6, 9 and 12 d postmortem of longissimus thoracis (LT) muscles, and corresponding average band intensity of sarcoplasmic (B) and myofibrillar (D) calpain-1. A mixture of supernatant from all muscles at 12 h post-mortem (taken as 100%) was used as a standard (st) for quantification across blots from different gels. All lanes were loaded with 15 μg of protein except the st lanes for myofibrillar fraction which were loaded with 3.7 μg of protein. The content of myofibril-bound calpain-1 was corrected to be comparable to the muscle content of 12 h sarcoplasmic calpain-1 using a myofibril : sarcoplasm protein ratio of 2 : 1. a-dWithin each trait, mean values with the different letter differ (P < 0.05).

a

b

cd

e e

0

30

60

90

120

0.5 1 3 6 9 12

Sarc

opla

smic

cal

pain

-2 (%

)

Storage (days)

(B)

(A)

Fig 2. Representative western blot (A) and average band intensity (B) of calpain-2 from sarcoplasmic protein fraction at 0.5 1, 3, 6, 9 and 12 d post-mortem of the longissimus thoracis (LT) muscles. A mixture of supernatant from all muscles at 12 h post-mortem were used as a standard (taken as 100%) for quantification across blots from different gels. All lanes were loaded with 15 μg of protein. a-dWithin each trait, mean values with the different letter differ (P < 0.05).

J. Lyu and P. Ertbjerg

Food Chemistry 372 (2022) 131347

4

3.3. Ca2+ titration of myofibril-bound proteases

The proteolytic potential of myofibril-bound proteases were inves-tigated on day 6 myofibrils as they had the greatest amount of bound calpain-1 (Fig. 1C). Myofibrils were isolated and Ca2+ titration curves were obtained in two separate assays. One assay observed the release of peptides from the myofibrils induced by myofbril-bound protease and another observed the desmin degradation. The amount of peptides released from the myofibrils increased with the increase of Ca2+ in two distinct concentration areas (Fig. 5A). The released peptides increased rapidly when Ca2+ concentration was increased from 0 to 0.06 mM, then

it was stable from 0.06 to 0.2 mM, followed by a pronounced increase from 0.4 to 0.8 mM. Desmin degradation showed a similar trend compared to that of peptide release (Fig. 5B and C). Desmin degradation increased rapidly with Ca2+ concentration up to 0.06 mM, then more slowly to 0.4 mM, followed by a pronounced degradation from 0.4 to 0.6 mM. The data indicates the presence of two distinct myofibril-bound proteolytic activities having Ca2+ requirements identical to that of calpain-1 and calpain-2, respectively.

4. Discussion

In the present study, we have shown that during postmortem storage of pork, the content of sarcoplasmic calpains decreased, while myofibril- bound calpain-1 increased until day 6 and thereafter decreased (Figs. 1 and 2). In parallel desmin degradation occurred (Fig. 3). During the entire storage period to day 12, isolated myofibrils contained sufficient proteases to degrade desmin upon activation by Ca2+ at a concentration corresponding to activation of calpain-1, and further degradation occurred at a concentration also activating calpain-2 (Fig. 4). Ca2+

titration curves demonstrated that day 6 myofibrils contained two distinct proteolytic activities corresponding to the known Ca2+ re-quirements for calpain-1 and calpain-2, respectively (Fig. 5). The rela-tive amount of desmin degraded was greater for the latter, suggesting that a greater amount of calpain-2 was bound to the myofibrils than that of calpain-1. However, we were not able to confirm this explanation by western blot towards calpain-2, probably due to lower sensitivity of the calpain-2 antibody relative to that of calpain-1.

Desmin is one of the most susceptible substrate of calpains (Bhat et al., 2018; Nelson & Traub, 1982). In the current study, there was almost no degradation products of desmin on day 1 (Fig. 3) and similar results have been reported in other studies (Huff-Lonergan et al., 1996; Kristensen & Purslow, 2001). The degree of desmin degradation was higher when myofibrils were incubated with Ca2+ than without Ca2+

and there was substantially more degradation products when myofibrils were incubated with 5 mM Ca2+ compared to that of 0.05 mM Ca2+

(Fig. 4). Zeng, Li, & Ertbjerg (2017) likewise observed that more desmin was degraded when myofibrils were incubated with higher Ca2+

e

d

c

b

a

0

20

40

60

80

100

120

1 3 6 9 12

Inta

ctde

smin

(%)

Storage time (days)

(B)

(A)

Fig 3. Western blot (A) and average band intensity (B) of desmin at 1, 3, 6, 9 and 12 d postmortem of the longissimus thoracis (LT) muscles. Desmin intensity at day 1 was taken as 100%. All lanes were loaded with 15 μg of protein. a-

eWithin each trait, mean values with the different letter differ (P < 0.05).

ax

bx

cx

dxdx

ay

by

cy dydy

az

bz cz dz dz0

20

40

60

80

100

120

1 3 6 9 12

Inta

ct d

esm

in (%

)

Storage time (days)

0 mM Ca0.05 mM Ca5 mM Ca

(B)

(A)

Fig 4. Representative western blot (A) and average band intensity (B) of desmin after incubation of myofibrils at 1, 3, 6, 9 and 12 d postmortem with different concentrations of Ca2+ (0, 0.05, and 5 mM). The standard (st) was a mixture of myofibrils from all muscles at 1d post-mortem (taken as 100%). All lanes were loaded with 10 μg of protein. a-d, x-zWithin Ca2+ concentrations (x-z) and storage days (a-d), mean values with different letter differ (P < 0.05).

J. Lyu and P. Ertbjerg

Food Chemistry 372 (2022) 131347

5

concentrations. In addition, the titration curves suggest that not only calpain-1, but also calpain-2 bound to myofibrils postmortem and the myofibril-bound calpains were able to degrade desmin upon activation with Ca2+. However, some studies reported that myofibril-bound cal-pain-1 had little activity, considering that a large amount of calpain-1 bound to myofibrils during storage (Boehm et al., 1998; Delgado, Geesink, Marchello, Goll, & Koohmaraie, 2001b). This may be because they used proteolysis of casein as an indicator to evaluate the activity of myofibril-bound calpain-1. It seems that myofibril-bound calpains cannot hydrolyze exogenous protein possibly due to steric hindrance of access to the active sites in myofibril-bound calpains.

Calpain-1 in the sarcoplasm decreased during storage, while myofibril-bound calpain-1 first increased to day 6 and then decreased (Fig. 1). Several studies have shown that the extractable activity of calpain-1 in bovine, ovine and porcine muscles declines substantially within the first one to two days postmortem. Delgado, Geesink, Mar-chello, Goll, & Koohmaraie (2001a) found that the extractable activity of calpain-1 on day 3 decreased to 19% of its initial activity in normal sheep longissimus dorsi muscle stored at 4 ◦C and was no longer detect-able after 10 days of storage. The activity of calpain-1 in beef stored at 4 ◦C decreased nearly to zero within 48 h postmortem (Camou et al., 2007). However, in present study, results of western blots showed that the content of sarcoplasmic calpain-1 decreased much slower than the reported extractable activity of calpain-1 during storage and there was still 33% calpain-1 in the sarcoplasm at the end of the storage (Fig. 1). The difference between extractable activity and sarcoplasmic content of calpain may due to that calpain loses its extractable activity because of instability of autolyzed calpain, and at least a part of it is still present in the sarcoplasm and can be recognized by its specific antibody. In agreement, in the study of (Rowe, Maddock, Lonergan, & Huff- Lonergan, 2004), western blot analysis of sarcoplasmic calpain-1 also showed that there was still a relatively large amount of autolyzed calpain-1 in beef after 15 days storage at 4 ◦C. Unlike the present study, where we quantified the combined native and autolyzed forms of calpain-1, Ilian, Bekhit, & Bickerstaffe (2004) only quantified the native

calpain-1 in the sarcoplasm during 7 days storage and they found that the content of native calpain-1 decreased from 50% on day 1 to nearly 0 on day 3 in lamb. We obtained a similar result that a significant pro-portion of calpain-1 was native on day 1 but autolyzed thereafter. Rowe et al. (2004) also found that native calpain-1 in beef was only present the first 2 days postmortem.

We have previously demonstrated a direct link between activation of calpain-2 by Ca2+ and its binding to myofibrils (Lyu & Ertbjerg, 2021). Loss of extractable activity of calpain, which has often been reported in literature especially in relation to calpain-1, can therefore be hypothe-sized to be the result of three distinct events i) binding of calpains to myofibrils and other subcellular organelles, ii) inhibition due to for-mation of a complex with calpastatin, and iii) instability caused by autolysis. Several studies have reported that calpain-1 gradually asso-ciated with myofibrils during postmortem storage (Boehm et al., 1998; Melody et al., 2004; Rowe et al., 2004). In agreement with our results, Delgado et al. (2001b) observed that the activity of myofibril-bound calpain-1 first increased, and then decreased during 10-day storage. It can be hypothesized that during storage calpains gradually bind to myofibrils induced by the increase of Ca2+ in the sarcoplasm, and thereafter they degrade structural proteins. A relative large change in calpain activity is observed during the initial one to three days post-mortem, and in agreement desmin is degraded faster in this period compared to later postmortem. The decreasing trend of myofibril-bound calpain-1 later postmortem is in agreement with the hypothesis that once myofibril-bound calpain completes the proteolytic degradation of available substrates in the vicinity of where it is bound in the myofibril structure, then calpain is released from the myofibrils to the sarcoplasm. The role of calpain-1 in ageing has been questioned by the fact that there is a time difference between the substantial decrease of extractable ac-tivity of calpain-1 early postmortem and degradation of myofibrillar proteins later. However, once calpain-1 binds to myofibrils, its extract-able activity will not be measured by in vitro assays and this is reflected as loss of extractable activity. Subsequently, calpain-1 may be active in the form of myofibril-bound calpain-1 (Figs. 4-5). It has been reported

0.6

0.8

1.0

1.2

1.4

1.6

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Prot

ein

cont

ent(

mg/

mlL

)

Ca2+ concentration (mM)

(A)

(B)

(C)

0

20

40

60

80

100

120

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Inta

ctde

smin

(%)

Ca2+ concentration (mM)

Fig 5. The relative protein content of supernatant (A), representative western blot of desmin in the myofibril fraction (B) and average band intensity (C) after incubation of myofibrils on day 6 with various Ca2+ concentrations at room temperature for 2 h. The standard (st) was a mixture of myofibrils from all muscles at 6 d post-mortem (taken as 100%). Each lane was loaded with 8 μg porcine skeletal muscle myofibrillar protein.

J. Lyu and P. Ertbjerg

Food Chemistry 372 (2022) 131347

6

that calpastatin cannot inhibit the proteolytic activity of myofibril- bound calpain-1 (Boehm et al., 1998; Delgado et al., 2001b). Thus, once calpain binds to myofibrils, its activity will not be affected by calpastatin. The effect of calpastatin on ultimate meat tenderness (Kemp, Sensky, Bardsley, Buttery, & Parr, 2010) is probably caused by its influence on the amount of calpain becoming bound to myofibrils.

It is generally believed that calpain-1 is activated early postmortem and has an important role in meat aging while the activity of calpain-2 is relatively stable during storage (Koohmaraie, Seidemann, Schollmeyer, Dutson, & Crouse, 1987; Veiseth, Shackelford, Wheeler, & Koohmaraie, 2001). However, decrease of calpain-2 activity during storage was re-ported in some studies (Boehm et al., 1998; Camou et al., 2007). In current study, a decreasing trend for sarcoplasmic calpain-2 content was observed similar to that of calpain-1 although the decrease was rela-tively slower, and the difference between the change of calpain-1 and calpain-2 diminished gradually in the later phase of storage (Figs. 1 and 2). The assays of incubation of myofibrils with Ca2+ and Ca2+ titration suggest that myofibril-bound calpain-2 also degraded structural proteins at higher concentration of Ca2+. These observations suggest that calpain-2 also contribute to the proteolysis of myofibrillar proteins, especially later postmortem as the free sarcoplasmic concentration be-comes higher. In our previous study, binding of exogenously added calpain-2 to myofibrils was induced by addition of Ca2+ and this myofibril-bound calpain-2 was proteolytically active to degrade desmin (Lyu & Ertbjerg, 2021). Combined with the present result, we speculate that calpain-2 undergoes a similar process as calpain-1, but later during storage, so that part of calpain-2 gradually binds to myofibrils with the increase of sarcoplasmic Ca2+ concentration later postmortem and then the myofibril-bound calpain-2 have proteolytic potential to degrade myofibrillar proteins.

5. Conclusion

The amount of sarcoplasmic calpain-1 and calpain-2 decreases with storage, while the content of myofibril-bound calpain-1 increases until day 6 postmortem, suggesting that the calpains translocate from sarco-plasm to myofibrils during postmortem storage. Desmin in myofibrils is degraded by associated proteases upon incubation with Ca2+ in the order 5 mM > 0.05 mM > 0. Ca2+ titration curves indicate that day 6 myofibrils contain two distinct proteolytic activities corresponding to the known Ca2+ requirements for calpain-1 and calpain-2, respectively. Taken together, the results indicate that both calpain-1 and calpain-2 become bound to myofibrils during storage and that the myofibril- bound proteases can be activated by Ca2+ to degrade desmin. Hence, myofibril-bound calpains could play an important role in the proteolysis process in meat during storage.

CRediT authorship contribution statement

Jian Lyu: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization, Funding acquisition. Per Ertbjerg: Conceptualization, Methodology, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors would like to thank the China Scholarship Council for financial support.

References

Bhat, Z. F., Morton, J. D., Mason, S. L., & Bekhit, A.-D. (2018). Role of calpain system in meat tenderness: A review. Food Science and Human Wellness, 7(3), 196–204.

Boehm, M. L., Kendall, T. L., Thompson, V. F., & Goll, D. E. (1998). Changes in the calpains and calpastatin during postmortem storage of bovine muscle. Journal of Animal Science, 76(9), 2415–2434.

Camou, J. P., Marchello, J. A., Thompson, V. F., Mares, S. W., & Goll, D. E. (2007). Effect of postmortem storage on activity of µ- and m-calpain in five bovine muscles. Journal of Animal Science, 85(10), 2670–2681.

Capetanaki, Y., Milner, D. J., & Weitzer, G. (1997). Desmin in Muscle Formation and Maintenance: Knockouts and Consequences. Cell Structure and Function, 22(1), 103–116.

Colle, M. J., & Doumit, M. E. (2017). Effect of extended aging on calpain-1 and -2 activity in beef longissimus lumborum and semimembranosus muscles. Meat Science, 131, 142–145.

Delgado, E. F., Geesink, G. H., Marchello, J. A., Goll, D. E., & Koohmaraie, M. (2001a). The calpain system in three muscles of normal and callipyge sheep. Journal of Animal Science, 79(2), 398–412.

Delgado, E. F., Geesink, G. H., Marchello, J. A., Goll, D. E., & Koohmaraie, M. (2001b). Properties of myofibril-bound calpain activity in longissimus muscle of callipyge and normal sheep. Journal of Animal Science, 79(8), 2097–2107.

Fritz, J. D., & Greaser, M. L. (1991). Changes in Titin and Nebulin in Postmortem Bovine Muscle Revealed by Gel Electrophoresis, Western Blotting and Immunofluorescence Microscopy. Journal of Food Science, 56(3), 607–610.

GOLL, D. E., Thompson, V. F., Li, H., Wei, W., & Cong, J. (2003). The calpain system. Physiological Reviews, 83(3), 731–801.

Huff-Lonergan, E., & Lonergan, S. M. (2005). Mechanisms of water-holding capacity of meat: The role of postmortem biochemical and structural changes. Meat Science, 71 (1), 194–204.

Huff-Lonergan, E., Mitsuhashi, T., Beekman, D. D., Parrish, F. C., Jr., Olson, D. G., & Robson, R. M. (1996). Proteolysis of specific muscle structural proteins by µ-calpain at low pH and temperature is similar to degradation in postmortem bovine muscle. Journal of Animal Science, 74(5), 993–1008.

Ilian, M. A., Bekhit, A.-D., & Bickerstaffe, R. (2004). The relationship between meat tenderization, myofibril fragmentation and autolysis of calpain 3 during post- mortem aging. Meat Science, 66(2), 387–397.

Kemp, C. M., Sensky, P. L., Bardsley, R. G., Buttery, P. J., & Parr, T. (2010). Tenderness – An enzymatic view. Meat Science, 84(2), 248–256.

Koohmaraie, M., Seidemann, S. C., Schollmeyer, J. E., Dutson, T. R., & Crouse, J. D. (1987). Effect of post-mortem storage on Ca++-dependent proteases, their inhibitor and myofibril fragmentation. Meat Science, 19(3), 187–196.

Kristensen, L., & Purslow, P. P. (2001). The effect of ageing on the water-holding capacity of pork: Role of cytoskeletal proteins. Meat Science, 58(1), 17–23.

Liu, J., Arner, A., Puolanne, E., & Ertbjerg, P. (2016). On the water-holding of myofibrils: Effect of sarcoplasmic protein denaturation. Meat Science, 119, 32–40.

Liu, J., Ruusunen, M., Puolanne, E., & Ertbjerg, P. (2014). Effect of pre-rigor temperature incubation on sarcoplasmic protein solubility, calpain activity and meat properties in porcine muscle. LWT - Food Science and Technology, 55(2), 483–489.

Lyu, J., & Ertbjerg, P. (2021). Ca2+-induced binding of calpain-2 to myofibrils: Preliminary results in pork longissimus thoracis muscle supporting a role on myofibrillar protein degradation. Meat Science, 172, 108364. https://doi.org/ 10.1016/j.meatsci.2020.108364

Melody, J. L., Lonergan, S. M., Rowe, L. J., Huiatt, T. W., Mayes, M. S., & Huff- Lonergan, E. (2004). Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles. Journal of Animal Science, 82(4), 1195–1205.

Nelson, W. J., & Traub, P. (1982). Purification and further characterization of the Ca2+- activated proteinase specific for the intermediate filament proteins vimentin and desmin. Journal of Biological Chemistry, 257(10), 5544–5553.

Pearce, K. L., Rosenvold, K., Andersen, H. J., & Hopkins, D. L. (2011). Water distribution and mobility in meat during the conversion of muscle to meat and ageing and the impacts on fresh meat quality attributes — A review. Meat Science, 89(2), 111–124.

Pomponio, L., & Ertbjerg, P. (2012). The effect of temperature on the activity of μ- and m- calpain and calpastatin during post-mortem storage of porcine longissimus muscle. Meat Science, 91(1), 50–55.

Rowe, L. J., Maddock, K. R., Lonergan, S. M., & Huff-Lonergan, E. (2004). Oxidative environments decrease tenderization of beef steaks through inactivation of μ-calpain. Journal of Animal Science, 82(11), 3254–3266.

Shackelford, S. D., Wheeler, T. L., Meade, M. K., Reagan, J. O., Byrnes, B. L., & Koohmaraie, M. (2001). Consumer impressions of Tender Select beef. Journal of Animal Science, 79(10), 2605–2614.

Taylor, R. G., Geesink, G. H., Thompson, V. F., Koohmaraie, M., & Goll, D. E. (1995). Is Z- disk degradation responsible for postmortem tenderization? Journal of Animal Science, 73(5), 1351–1367.

Veiseth, E., Shackelford, S. D., Wheeler, T. L., & Koohmaraie, M. (2001). Effect of postmortem storage on µ-calpain and m-calpain in ovine skeletal muscle. Journal of Animal Science, 79(6), 1502–1508.

Zeng, Z., Li, C., & Ertbjerg, P. (2017). Relationship between proteolysis and water- holding of myofibrils. Meat Science, 131, 48–55.

Zhang, Y., & Ertbjerg, P. (2018). Effects of frozen-then-chilled storage on proteolytic enzyme activity and water-holding capacity of pork loin. Meat Science, 145, 375–382.

J. Lyu and P. Ertbjerg