Combined effect of epinephrine and exercise on calpain/calpastatin and cathepsin B and L activity in...

P. Ertbjerg, P. Henckel, A. Karlsson, L. M. Larsen and A. J. Mollerand L activity in porcine longissimus muscle

Combined effect of epinephrine and exercise on calpain/calpastatin and cathepsin B

1999, 77:2428-2436.J ANIM SCI

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1The authors thank L. Jørgensen for technical assistance. Thiswork was supported by the Danish Ministry of Food, Agricultureand Fisheries and the Federation of Danish Pig Producers andSlaughterhouses.

2To whom correspondence should be addressed (Phone: +45 3528 32 55; fax: +45 35 28 33 41; E-mail: [email protected]).

Received July 20, 1998.Accepted February 8, 1999.

Combined Effect of Epinephrine and Exercise on Calpain/Calpastatin andCathepsin B and L Activity in Porcine Longissimus Muscle1

Per Ertbjerg*,2, Poul Henckel‡, Anders Karlsson‡, Lone M. Larsen†,and Anders J. Møller*

*Department of Dairy and Food Science, †Chemistry Department, Royal Veterinary and AgriculturalUniversity, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark and

‡Danish Institute of Agricultural Sciences, Department of Animal Product Quality,Research Centre Foulum, DK-8830 Tjele, Denmark

ABSTRACT: The objective of the study was toimprove the understanding of the relationship be-tween the effect of epinephrine plus exercise and meattenderness. The calpain, calpastatin, and cathepsin B+ L activities and postmortem proteolysis in porcinelongissimus muscle were studied. The muscle glycogenstores were depleted in five pigs by s.c. injection ofepinephrine (.3 mg/kg) at 15 h antemortem andexercise on a treadmill (5 min, 3.8 km/h) immediatelybefore slaughter. Antemortem injection of epinephrineand treadmill exercise resulted in higher ultimate pH(6.32 vs 5.66 in control) and decreased ( P < .05) thawloss, cooking loss, and shear force values. The muscleenergy depletion treatment increased ( P < .05) themuscle m-calpain activity measured 42 min postmor-tem, and at 24 h m-calpain activity was still approxi-mately 50% greater in the high ultimate pH group.Also, as the ratio of m-calpain to calpastatin increased( P < .01), the overall proteolytic potential of thecalpain system were greater. These observations

suggest that the muscle energy level may influencethe activity of the calpain system in the living animal.The high ultimate pH group showed lower ( P < .001)cathepsin B + L activity in the myofibrillar and thesoluble fractions after 8 d of storage, suggesting thatthe increased ultimate pH increased the stability ofthe lysosomal membrane and thereby reduced therelease of cathepsins from the lysosomes duringstorage. The SDS-PAGE showed increased ( P < .001)degradation of a 39-kDa band in the epinephrine andexercise-treated samples. Degradation products at 30,31, and 32 kDa were labeled by troponin-T antibody inwestern blot. An appearing 24-kDa band was identi-fied as a troponin-I degradation product in westernblot. The proteolytic degradation pattern of myofibril-lar proteins during storage differed in control andtreated samples, supporting the hypothesis that cal-pain-mediated proteolysis was affected after treat-ment, resulting in meat with high ultimate pH.

Key Words: Pigs, Calpain, Cathepsins, Epinephrine, Proteolysis, Tenderness

1999 American Society of Animal Science. All rights reserved. J. Anim. Sci. 1999. 77:2428–2436

Introduction

Postmortem ( PM) muscle acidification is mainlydependent on the available glycogen stores at slaugh-ter. Ultimate pH values around 5.6 are normal inlongissimus muscle ( LM) , but pH values above 6.2were reported in porcine (Fernandez et al., 1994;Henckel et al., 1997), ovine (Watanabe and Devine,

1996; Watanabe et al., 1996), and bovine (Yu andLee, 1986; Geesink et al., 1992; Beltran et al., 1997)muscles after muscle glycogen stores were depleted byepinephrine injection, epinephrine injection and sub-sequent exercise, or stress before slaughter. Highultimate pH has been associated with increased meattenderness, possibly arising from increased water-holding properties producing swelling (Offer andTrinick, 1983; Tornberg, 1996).

Additionally, high ultimate pH favors the action ofproteolytic enzymes with optimal activity in theneutral pH range. Yu and Lee (1986) found that highpH resulted in increased degradation of troponin Tand removal of a-actinin and Z-line. Similar changeswere observed after incubation of isolated myofibrilswith m-calpain at high pH (Koohmaraie et al., 1986).More rapid degradation of high molecular weight



PROTEOLYTIC CHANGES IN ENERGY-DEPLETED PIG MUSCLE 2429

proteins in high-pH meat may be related to calpainactivity in beef (Geesink et al., 1992) and sheep(Watanabe and Devine, 1996).

There is little available information on the effect ofepinephrine on proteolytic enzyme activities in porcinemuscle. Sensky et al. (1996) infused pigs i.v. withepinephrine for 1 wk but reported no effect on m- andm-calpain activity at slaughter. Changes in tendernessand in myofibrillar proteins during storage were notconsidered. Our objectives were to study the effects ofdepleting muscle glycogen stores by epinephrineinjection 15 h antemortem and treadmill runningbefore slaughter on meat tenderness as well as oncalpain, calpastatin, and cathepsin activity and on PMproteolysis in porcine LM.

Materials and Methods

Animals and Treatment

The animals used in this study were 10 crossbredfemale pigs (Duroc sire and Large White-Landracedam) from five litters. All parents were halothanetested to exclude carriers of the halothane gene fromthis population. Pigs were slaughtered by CO2 stun-ning at an experimental station at approximately 90kg of live weight. The muscle glycogen stores weredepleted in five pigs by s.c. injection of epinephrine (.3mg/kg) at 15 h antemortem and exercise on atreadmill (5 min, 3.8 km/h) immediately beforeslaughter. The combined epinephrine and exercisetreatment were to ensure a more complete depletion ofmuscle energy stores at time of slaughter as describedby Henckel et al. (1997). Five siblings to the treatedanimals served as controls, and at 15 h antemorteminjections of saline solution were given.

Sampling Procedure

For determination of the calpain system, a sample(3 cm × muscle diameter) from LM was excised at the15th rib 35 min and at the 14th rib 24 h PM. Thesamples were vacuum packed after excision, frozen inliquid nitrogen, and stored at −80°C until analysis.Samples were analyzed within 4 wk after frozenstorage. The sample excised at 35 min was vacuumpacked at an ambient temperature of 15°C and frozenat 42 min PM. At 24 h PM, a section of LM wasexcised at the 8th to the 13th rib, and subsampleswere vacuum packed. The section at the 13th rib wasused for cathepsin B + L measurements. One subsam-ple was processed immediately after excision at 24 h,and another was vacuum packed and processedwithout freezing after 8 d of storage. The remainingLM section was subdivided into two equal parts usedfor thaw loss, cooking loss, Warner-Bratzler shearforce, and SDS-PAGE. One part was vacuum packedand then frozen at −20°C (24 h sample), and the other

was stored at 2°C until 8 d PM before vacuum packingand freezing.

pH Determination

The pH of the LM was determined with a pH meter(Metrohm Model 704, Switzerland) equipped with ainsertion glass electrode (Hamilton Tiptrode P/N: 238’080, Switzerland) calibrated in buffers at pH 4.01 and7.00 (Radiometer, Copenhagen, Denmark). TriplicatepH measurement of each muscle was performed at 45min and at 24 h PM. The 24-h measurement was usedas an indication of ultimate pH.

Thaw Loss, Cooking Loss, and Shear Force

Thaw loss, cooking loss (% wt/wt) and shear force( N ) were determined on the same sample (approx.200 g). To determine thaw loss, the vacuum bags wereweighed and opened, excess liquid was removed fromthe bags, and the samples were reweighed. Thensamples were vacuum packed again and heated for 60min at 80°C. Cooking loss was determined by remov-ing excess liquid after cooking and weighing thesamples again. Warner-Bratzler shear force measure-ments were performed as described by Møller (1981).Each sample resulted in 12 shear force deformationcurves for calculating mean values for max shearforce.

Separation of Calpains and Calpastatin

Calpains were separated by modification of themethod described by Iversen et al. (1993). All stepswere carried out at 4°C. Muscle samples were takenfrom the freezer and allowed to thaw at 4°C for 1 hbefore being finely chopped. Ten grams of muscle werehomogenized (Ultra Turrax Mixer T 45, 4 × 30 s at 1/2maximal speed and 2 × 30 s at 3/4 maximal speed, 30 scooling between bursts) in six volumes of buffer (50mM Tris·HCl, 5 mM EDTA, 10 mM monothioglycerol,150 nM pepstatin A, pH 8.0). The homogenate waskept on ice for 15 min and then centrifuged (30,000 ×g, 15 min, 4°C), and the supernatant was filteredthrough a .20-mm filter (Sartorius, Gottingen, Germa-ny). One milliliter of the extract was used fordetermination of calpastatin activity (see below). Tenmilliliters of the extract were loaded on a mono-Q HR5/5 column (Pharmacia Biotech, Uppsala, Sweden)and m-calpain was isolated and measured as describedbelow. Using this procedure, calpastatin was found toelute in two peaks, with the second peak coelutingwith m-calpain. To determine m-calpain, a hydrophobiccolumn was used to separate calpains from calpasta-tin. Twenty milliliters of the filtered supernatant wasmade 300 mM with NaCl (from a 5 M solution) andloaded onto a phenylsepharose CL-4B column (1.2 × 8cm, 1 mL/min; Pharmacia Biotech), previouslyequilibrated with buffer A (20 mM Tris·HCl, 300 mMNaCl, 5 mM EDTA, 10 mM monothioglycerol, 2.5 mM



ERTBJERG ET AL.2430

NaN3, pH 7.5). The column was washed with buffer Auntil the absorption (A278) reached the baseline.Calpains were eluted with buffer B (20 mM Tris·HCl,5 mM EDTA, 10 mM monothioglycerol, 2.5 mM NaN3,pH 7.5). Twenty milliliters of the calpain-containingeluate was loaded on a mono-Q HR 5/5 (PharmaciaBiotech, Uppsala, Sweden) column previouslyequilibrated with buffer B. Loading and eluting ratewas 1 mL/min. The column was washed with buffer Buntil the absorption (A278) reached the baseline.Within 26 min, a clear separation of m-calpain and m-calpain was obtained by a stepped gradient to .6 MNaCl using buffer A: 0 to 2 mL (0 to 84 mM NaCl), 2to 9 mL (84 to 93 mM NaCl), 9 to 11 mL (93 to 300mM NaCl), 11 to 16 mL (300 mM NaCl), 16 to 20 mL(300 to 600 mM NaCl), and 20 to 26 mL (600 mMNaCl). m-Calpain (eluted at approx. 13 min) and m-calpain (eluted at approx. 20 min) were each collectedin a 3-mL fraction. The recovery of m-calpain at thephenylsepharose column was estimated by calculatingthe recovery of m-calpain; m-calpain in each samplewas determined in the 10-mL extract directly loadedonto a mono-Q column and in the 20-mL extractloaded first on phenylsepharose and then on mono-Qcolumn.

Calpain Activity Measurements

Calpain activity was determined using casein assubstrate and the method described by Iversen et al.(1993) but with volumes scaled down to 1/5. From thecalpain fraction, 100 mL were incubated with 300 mLincubation medium (100 mM Tris base, 10 mMmonothioglycerol, 1.0 mM NaN3, 5 mg/mL casein and5.0 mM CaCl2, pH adjusted to 7.5 with 1 M aceticacid). After 20 min incubation at 25°C, the reactionwas stopped by addition of 400 mL of 10% trichloroa-cetic acid, and tubes were centrifuged at 20,000 × g for3 min. One unit of calpain activity was defined as anincrease in absorbance at 278 nm of 1.0 per hour at25°C. Results were corrected for noncalcium-depen-dent proteolytic activity by using 10 mM EDTAinstead of CaCl2 in the incubation medium.

Calpastatin Determination

The 1 mL sample from the homogenate extractremoved for calpastatin determination was heated at100°C for 3 min and centrifuged at 20,000 × g for 4min. The calpastatin activity in the supernatant wasdetermined using various dilutions in the above-described casein assay containing .2 unit of m-calpainextracted and fractionated from other samples. Thevolume of heated supernatant sample never exceeded20 mL in the assay; the volume was 400 mL before theaddition of trichloroacetic acid. Incubation time was40 min at 25°C. From observation of the decrease inabsorbance at 278 nm, an inhibition curve wasgenerated. The linear part of this curve was used to

calculate calpastatin activity. One unit of calpastatinactivity was defined as the amount that inhibited oneunit of m-calpain activity.

Cathepsin B + L Activity Measurementsin Subcellular Fractions

Cathepsin B + L activities were determined onnonfrozen samples by a slight modification of themethod described by Ertbjerg et al. (1999). Allprocedures were carried out at 0 to 4°C. Twomilliliters per gram of tissue of homogenization buffer(100 mM sucrose, 100 mM KCl, 50 mM Tris·HCl, 10mM sodium pyrophosphate, 1 mM Na2EDTA, pH 7.2)containing 50 mg/mL nagarse (Sigma Chemical, St.Louis, MO, protease Type XXVII) dissolved immedi-ately before use were added to approximately 1.5 g oftissues. The suspension was finely minced withscissors and then incubated 5 min. Then thehomogenization buffer (20 mL/g tissue) withoutnagarse was added. Homogenization was performedusing a motor-driven Potter-Elvehjem-typehomogenizer (glass/Teflon, clearance .15 mm) bymaking 10 complete passes at 1,500 rpm. Thehomogenate was filtered through cheesecloth. Thefiltrate was centrifuged at 1,100 × g for 10 min toobtain a myofibrillar fraction. The supernatant wasthen centrifuged at 20,000 × g for 20 min to obtain alysosomal fraction. Finally, the supernatant wascentrifuged at 100,000 × g for 60 min to obtain amicrosomal fraction (pellet) and a soluble fraction(the final supernatant). The myofibrillar fraction wasresuspended in 5 mL and the other pellets in 2 mL ofbuffer (85 mM Na acetate, 15 mM acetic acid, 1 mMNa2EDTA, pH 5.5) and rapidly frozen in liquidnitrogen. The combined cathepsin B + L activitieswere determined fluorimetrically according toKirschke et al. (1983) using the common substrate N-CBZ-L-phenylalanyl-L-arginine-7-amido-4-methylcou-marin (Z-Phe-Arg-NMec). Activities of cathepsin B +L are expressed as mU (the release of 1 nmol ofproduct per minute).

Isolation of Myofibrils

Myofibrils were prepared as previously described(Møller et al., 1973) and protein concentration wasdetermined with the biuret procedure (Gornall et al.,1949) using BSA as standard. Samples were preparedfor SDS-PAGE by dissolving myofibrils in samplebuffer (62.5 mM Tris·HCl [pH 6.8], 2% [wt/vol] SDS,10% [vol/vol] glycerol, 320 mM dithiothreitol, and.0025% [wt/vol] bromophenol blue). Samples wereheated at 100°C for 4 min before they were loadedonto gels.

SDS-PAGE Analysis

Electrophoretic procedures of Laemmli (1970) werefollowed. Each sample was run in duplicate using




Table 1. Thaw loss, cooking loss, and shear force at1 and 8 d postmortem (PM) as affected by

epinephrine and exercise treatmenta

aData are expressed as mean ± SE.bBased on Student’s t-test for unpaired data.

Control TreatedTrait Time PM n = 5 n = 5 P-valueb

Thaw loss, % 1 d 6.33 ± .38 1.44 ± .44 <.0018 d 6.67 ± 1.30 .78 ± .24 <.01

Cooking loss, % 1 d 26.0 ± .41 19.8 ± 1.73 <.018 d 27.1 ± .76 17.9 ± 1.77 <.01

Shear force, N 1 d 44.1 ± 2.63 25.3 ± 4.44 <.018 d 32.6 ± 2.50 20.7 ± 3.26 <.05

Tris-glycine precast gels (Novex, San Diego, CA)under conditions as described by the supplier on aXCell II unit (Novex). Myofibrillar proteins wereseparated using 1 mm thick 14% acryl amide slab gelswith 2.6% (wt/vol) bisacryl amide. The amount ofprotein loaded onto each lane was 40 mg. The gels wererun at a constant voltage setting of 125 V forapproximately 90 min at 25°C. After electrophoresis,gels were either stained for visualization of proteinbands or were transferred by electroelution to nitrocel-lulose membranes. Gels used for examination of allprotein bands were stained for 3 h in an excess of asolution containing .1% (wt/vol) Coomassie brilliantblue R-250, 40% (vol/vol) ethanol, and 7% glacialacetic acid. Gels were destained in an excess of thesame solution without the Coomassie brilliant blue.Densitometric scans of gels were performed using aCREAM scanning system (Kem-En-Tec, Copenhagen,Denmark). To standardize image analysis procedures,the baseline was defined with the computer software(low-level background correction). Peak area values ofSDS-PAGE bands were determined and expressed aspercentage of the actin band in the sample.

Western Blotting

Gels were briefly rinsed with transfer buffer (12mM Tris, 96 mM glycine and 20% methanol [vol/vol]).Protein bands were blotted onto nitrocellulose mem-branes using a XCell II Blot Module (Novex) at aconstant voltage setting of 25 V for 90 min at 25°C.Membranes were then incubated for 1 h at 25°C inblocking solution (80 mM disodium hydrogen or-thophosphate, 100 mM sodium chloride, .1% [vol/vol]polyoxyethylene sorbitan monolaurate [Tween-20],and 5% [wt/vol] nonfat dry milk). Primary antibodiesused in the Western blotting procedures includedmonoclonal anti-vinculin (clone hVIN-1, Sigma Chem-ical), diluted 1:10,000 in PBS-Tween (blocking solu-tion without nonfat dry milk) containing 1% nonfatdry milk; monoclonal anti-troponin-T (JLT-12, SigmaChemical) diluted 1:20,000 in PBS-Tween containing1% nonfat dry milk; and monoclonal anti-troponin-I(MAB 1691, Chemicon) diluted 1:50,000 in PBS-Tween containing 1% nonfat dry milk. Incubation withprimary antibodies was for 1 h at 25°C. Blots werewashed three times, 15 min per wash, in PBS-Tween.Bound primary antibodies were labeled with goat-antimouse IgG horseradish peroxidase-conjugatedsecondary antibodies (NIF 825, Amersham, ArlingtonHeights, IL), diluted 1:5,000 in PBS-Tween, for 60min at 25°C. Blots were washed in PBS-Tween fourtimes, 15 min per wash, before detection. Achemiluminescent system was used to detect labeledprotein bands as described by the supplier (Amer-sham).

Statistical Analysis

The data were analyzed using two-way analysis ofvariance to determine main effects of storage time and

treatment on measured traits. Single mean valueswere compared using Student’s t-test for unpaireddata.

Results and Discussion

Thaw Loss, Cooking Loss, and Tenderness

The epinephrine and exercise ( EE) treatment todeplete the energy stores resulted only in a minor pHdifference at 45 min PM (6.58 vs 6.44 in control, P <.05), whereas the ultimate pH was substantial higher(6.32 vs 5.66, P < .01). At 1 and 8 d PM, mean thawloss and cooking loss were significantly less in samplesfrom EE treated animals (Table 1). Storage time didnot affect thaw loss ( P = .98) and cooking loss ( P =.76). The increased ultimate pH after EE treatmentwould be expected to improve the water-holdingcapacity. Improved meat water holding may, there-fore, explain the reduced thaw loss and cooking lossafter treatment. The EE treatment resulted at 1 d PMin significantly more tender meat (Table 1). Shearforce values of EE treated animals were 43% lower ( P< .01) at 1 d PM and were 36% lower ( P < .05) afterstorage for 8 d. In principle, the reduced toughness ofthe high ultimate pH group can be attributed eitherdirectly to the increased water-holding capacity (e.g.,water dilution of load bearing muscle elements afterlateral swelling) or to increased muscle proteolysis. Toevaluate the latter factor, we measured the activitiesof muscle proteolytic enzymes generally associatedwith tenderization.

Calpains

Table 2 shows that among the components of thecalpain system, only m-calpain activity was signifi-cantly affected by the muscle energy-depletion treat-ment. At 42 min and 24 h PM, m-calpain activitieswere 45 to 50% greater in the high ultimate pH group.In the caseinolytic assay, calpain activities expressedas units per gram muscle are by definition dependenton the assay volume. Taking this into account, we



ERTBJERG ET AL.2432

Table 2. Calpain-system activity (U/g muscle) at 42 min and 24 h postmortem(PM) as affected by epinephrine and exercise treatmenta

aData are expressed as mean ± SE.bBased on Student’s t-test for unpaired data.cNS = not statistically significant.


m-Calpain, U/g muscle 42 min 3.84 ± .53 5.56 ± .11 <.0524 h 2.46 ± .37 3.68 ± .32 <.05

m-Calpain, U/g muscle 42 min 9.47 ± .65 8.87 ± .47 NSc

24 h 8.44 ± .23 8.49 ± .46 NSCalpastatin, U/g muscle 42 min 13.8 ± .76 13.2 ± 1.5 NS

24 h 12.6 ± .49 11.0 ± 1.9 NSm-Calpain:calpastatin 42 min .28 ± .03 .42 ± .02 <.01

24 h .19 ± .02 .33 ± .01 <.001

found that the values obtained for m-calpain activitywere in the same range as values measured previouslywith the caseinolytic assay in porcine LM (Kooh-maraie et al., 1991b). However, the values obtainedfor m-calpain were not completely comparable becausewe found a m- to m-calpain ratio of approximately 1 to2 at 42 min PM, and Koohmaraie et al. (1991b) founda ratio of 1 to .75 on samples taken immediately afterslaughter. Using a commercially available m-calpainpreparation originating from porcine erythrocytes(Chemicon, Temecula, CA) as a standard, the meanextractable activity in the control LM at 42 min PMgiven in Table 2 was calculated to correspond toapproximately 30 mg of m-calpain per gram of muscle.

The measured increase in m-calpain activity afterepinephrine injection differed from earlier results froman experiment with beef, in which epinephrine injec-tion had only minor effects on m-calpain activity in LMmuscle (Geesink et al., 1992). In pigs, i.v. infusion ofepinephrine for a period of 1 wk showed no effect on m-and m-calpain in LM at time of slaughter (Sensky etal., 1996). During PM storage, it was reported that m-calpain and (less pronounced) m-calpain showedhigher activities after epinephrine infusion, but the m-calpain activity in their study decreased with storage.Therefore, the authors suggested that porcine LM m-calpain is much less stable than m-calpain in tissuesof ruminants. In contrast, in the present study,analysis of variance showed that m-calpain activity inpig LM was not significantly affected by storage ( P =.16) because storage only decreased m-calpain activity( P < .01) and calpastatin activity ( P < .05). Thispattern agrees with studies on beef muscle, for whichit was reported that during PM storage m-calpain andcalpastatin decreased while m-calpain remainednearly constant (Ducasting et al., 1985; Koohmaraieet al., 1987, 1995; O’Halloran et al., 1997).

Several studies have suggested that m-calpaincauses many of the proteolytic degradations of my-ofibrillar proteins in PM muscle and that this, in turn,is related to tenderization (e.g., Koohmaraie et al.,1986; Huff-Lonergan et al., 1996; Koohmaraie, 1996).

If m-calpain is not activated during storage (e.g., ifthe rise in intracellular free Ca2+ is insufficient toactivate m-calpain), then the m-calpain to calpastatinratio is likely to reflect the proteolytic potential of thecalpain system. The proteolytic potential of thecalpain system (taken as the above ratio) increasedsignificantly after the EE treatment (Table 2). At 45min PM, the pH of samples from treated animals was.14 pH units higher than controls. The increase in m-calpain activity and proteolytic potential at 42 minPM could have been influenced by differences inmuscle pH. Alternatively, the epinephrine injectionmay have induced antemortem changes in the calpainsystem. In that case, the proteolytic potential of thecalpain system is increased either by direct action ofthe hormone or indirectly, for example, by low muscleglycogen levels. Physiologically, low muscle glycogenlevels are found in the fasted state or after heavyexercise or animal fighting. In these situations,breakdown of muscle proteins provides amino acids forgluconeogenesis and energy metabolism. The calpainsystem has been suggested to initiate turnover ofmyofibrillar proteins by making specific cleavages thatrelease thick and thin filaments from the surface ofthe myofibril (Goll et al., 1989; Goll et al., 1992). Inthe present experiment, the observation that m-calpainactivity and calpain proteolytic potential increased inenergy-depleted pigs indicates that the muscle energylevel may influence the activity of the calpain systemin the living animal. We therefore propose that lowenergy stores in vivo lead to increased calpain-inducedproteolysis in pig muscle and promote mobilization ofproteins for gluconeogenesis and energy metabolism.Epinephrine stimulates b-adrenergic receptors (Mers-mann, 1989). In the literature, there are someindications that b-adrenergic receptor stimulationinfluences calpain activity. Iizuka et al. (1991)reported that calpain in rat heart was activated by b-adrenergic receptor stimulation under hypoxia. Syn-thetic b-adrenergic agonists are known to be able toincrease muscle growth. These compounds sup-plemented in the diet have been shown to affect the




Table 3. Cathepsin B + L activity (U/g muscle) in subcellular fractions at 1 and 8d postmortem (PM) as affected by epinephrine and exercise treatmenta

aData are expressed as mean ± SE.bBased on Student’s t-test for unpaired data.cNS = not statistically significant.


Myofibrillar 1 d 4.08 ± .72 3.71 ± .59 NSc

8 d 4.34 ± .23 2.71 ± .24 <.001Lysosomal 1 d 16.1 ± 1.73 16.7 ± 2.08 NS

8 d 16.2 ± 1.50 17.1 ± 2.60 NSMicrosomal 1 d 2.61 ± .43 2.33 ± .49 NS

8 d 2.23 ± .14 3.12 ± .79 NSSoluble 1 d .93 ± .03 1.04 ± .18 NS

8 d 1.50 ± .06 1.01 ± .08 <.001

calpain system of sheep and steers (Koohmaraie et al.,1991a, 1996; Koohmaraie and Shackelford, 1991;Wheeler and Koohmaraie, 1992; Pringle et al., 1993).In the majority of these studies, administration of b-adrenergic agonists for weeks resulted in an increasedlevel of calpastatin and in decreased calpain proteo-lytic potential (again defined as m-calpain to calpasta-tin ratio) after slaughter and increased meat tough-ness after aging. It is unclear why the effect of long-term feeding with synthetic b-adrenergic agonists oncalpain proteolytic potential and meat tenderness areopposite to the effect of epinephrine injection 15 hbefore slaughter (and any additional effect of tread-mill running) as described in the present study.Possible causes could be related to factors such asacute vs chronic effects, differences in receptor subtypebinding, receptor desensitization (down regulation),and differences between species, which are known toinfluence the response of adrenergic agonists (Mers-mann, 1989).

Lysosomal Enzymes

The effect of energy depletion treatment on thecellular distribution of the lysosomal enzymes cathep-sin B and L was also investigated. Table 3 shows thattreatment did not significantly affect cathepsin B + Lactivity in the four subcellular fractions at 1 d PM.After 8 d of storage, the cathepsin B + L activity in themyofibrillar and the soluble fraction was less ( P <.001) in the treated samples. This difference seemedto arise due to decreased activity in treated samples(myofibrillar fraction) or increased activity in controls(soluble fraction) during storage. Storage of beef hasbeen shown to increase free (soluble fraction) cathep-sin B + L activity (O’Halloran et al., 1997; Ertbjerg etal., 1999). Increasing pH from 5.5 to 6.0 in beefmuscle resulted in decreased free (soluble) lysosomalenzyme activity (Ertbjerg et al., 1999). In the presentexperiment with pork, the increase in ultimate pHafter glycogen depletion is likely to have increased the

stability of the lysosomal membrane. Reduced releaseof cathepsins from the lysosomes during storage may,therefore, explain the lesser cathepsin B + L activitiesin the soluble fraction in high ultimate pH samples.Cathepsin B and L are irreversibly denatured atneutral pH values, and this could contribute to theobserved low cathepsin B + L activity in the myofibril-lar and soluble fraction of treated samples at d 8.

Electrophoresis and Western Blots

Figure 1a shows representative SDS-PAGE profilesof extracted myofibrillar proteins. During storage from1 to 8 d, the most pronounced visible changes incontrols were appearance of a band at 24 kDa andbands at 30, 31, and 32 kDa. Storage of samples fromepinephrine- and exercise-treated animals did notresult in generation of the 24-kDa band to the sameextent (Figure 1a). Also, the 30-kDa band seemed tostain more strongly and perhaps with a tendency toless staining of the 31-kDa band. Semiquantitativedata on electrophoretic changes were obtained bydensitometric scanning of gels. Table 4 confirms thatthe 24-kDa band ( P < .001) and the 31-kDa band ( P <.05) did not increase during storage to the same levelin treated samples as seen in controls. After 8 d ofstorage, a band at 39 kDa showed decreased ( P <.001) intensity in treated samples (Table 4). Tropo-nin-T and tropomyosin migrate in this area (Ho et al.,1994). In beef LM, troponin-T degradation duringstorage resulted in the appearance of a 30-kDa band,whereas a 31-kDa band migrating slightly slower thanthe 30-kDa protein was not labeled by the troponin-Tantibody (Ho et al., 1994). This could indicate thatthe 30-kDa band appearing more pronounced in EEsamples in the present study (Table 4) was adegradation product of troponin-T. This was furtherinvestigated by western blotting using a monoclonalantibody against troponin-T (Figure 1c). The tropo-nin-T antibody labeled two bands at 37 and 39 kDa in1-d samples. These bands are likely to correspond to a



ERTBJERG ET AL.2434

Figure 1. Electrophoretically separated myofibrillar proteins from two control (lane 1, 2, 5, and 6) and twoepinephrine- and exercise-treated (lane 3, 4, 7, and 8) animals. Samples were stored for 1 d (lane 1 to 4) or 8 d (lane 5to 8). (a) Coomassie-stained gel. Ac = actin. Molecular weight markers (kDa) are indicated at the left side. (b) Westernblot using monoclonal vinculin antibody. Arrow at the left side designates the position of intact vinculin at 220 kDa.Two open triangles at the right side designates the position of two major degradation products. (c) Western blotusing monoclonal troponin-T antibody. Two arrows at the left side designates the position of two stained forms oftroponin-T at 39 and 37 kDa. Two open triangles at the right side designates the position of two major degradationproducts at 30 and 32 kDa. A third open triangle points at lane 1 at a 31 kDa band. (d) Western blot using monoclonaltroponin-I antibody. Arrow at the left side designates the position of intact troponin-I at 25 to 26 kDa. Open triangleat the right side points at degradation product at 24 kDa.

doublet band reported to be labeled by the sameantibody in 0-d samples in beef muscle (Huff-Lonergan et al., 1996). The 39-kDa band in thepresent study was to some extent degraded in8-d samples, and increased staining was observed inthe 30- to 32-kDa area. It would, therefore, seem thatnot only the 30-kDa band, but also the 31- and32-kDa bands generated in porcine LM containdegradation products from troponin-T. Control sam-ples seemed to be more labeled in the 31-kDa areathan EE samples, confirming the observation from theCoomassie stained gels that labeling in 30- to32-kDa area differed in control and EE samples. Ho etal. (1994) also reported that the troponin-I bandmigrated just below myosin light chain 1, that theamount of intact troponin-I decreased during storage,and then migrated at slightly lower molecular weight.Comparing these results with the electrophoretic

patterns in Figure 1a, it follows that the 26-kDa bandis likely to be composed of poorly resolved myosin lightchain 1 and troponin-I, with troponin-I migrating asthe lower component of the band. The 26-kDa bandwas partly degraded in control and EE-treated sam-ples during storage (Table 4), suggesting that tropo-nin-I was degraded to the 24-kDa band in controls andto lower molecular weight component(s) in EE-treated samples. Western blotting using a monoclonalantibody against troponin-I (Figure 1d) showed thattroponin-I antibody labeled a band at 25 to 26 kDa(arrow) in 1-d samples. This band migrated slightlyfaster to 24 kDa in 8-d control samples. Even thoughthe troponin-I band was extensively degraded in EEsamples, the 24-kDa band was not generated to thesame extent as in controls. These results, therefore,support the observations from the Coomassie stainedgels. Western blot using antibody against vinculin




Table 4. Density of SDS-PAGE bands relative toactin (%) at 1 and 8 d postmortem (PM) as

affected by epinephrine and exercise treatmenta

aData are expressed as mean ± SE.bBased on Student’s t-test for unpaired data.cNS = not statistically significant; NM = not measurable.

Control TreatedBand Time PM n = 5 n = 5 P-valueb

39 kDa 1 d 30.1 ± 1.3 30.8 ± 1.1 NSc

8 d 27.3 ± 1.2 19.5 ± .4 <.00137 kDa 1 d 28.1 ± 1.9 27.7 ± .8 NS

8 d 34.5 ± .68 29.9 ± 1.4 <.0532 kDa 1 d NM NM

8 d 4.1 ± .80 3.6 ± .7 NS31 kDa 1 d NM NM

8 d 5.5 ± 1.3 1.5 ± .3 <.0530 kDa 1 d 1.8 ± .4 1.9 ± .2 NS

8 d 4.2 ± .7 7.6 ± 2.3 NS26 kDa 1 d 32.3 ± 1.8 31.8 ± .2 NS

8 d 20.9 ± 1.7 21.5 ± 1.2 NS24 kDa 1 d NM NM

8 d 12.4 ± 2.2 2.9 ± 1.1 <.00119 kDa 1 d 26.2 ± 1.0 25.1 ± 1.1 NS

8 d 29.4 ± 2.0 26.4 ± 2.7 NS

labeled a band at approximately 220 kDa moststrongly in 1-d samples (Figure 1b). This bandseemed to be degraded to two major bands in8-d control samples. The faster migrating degradationproduct tended to split into a doublet in EE samples.The results show that the proteolytic degradationpattern of myofibrillar proteins differed in control andEE-treated samples. m-Calpain is known to haveoptimum in vitro activity at neutral pH, and theextractable amount increased in EE-treated samples(Table 2). Consequently, calpain-mediated proteolysiswould be expected to be increased in the high pHsamples. This is likely to be part of the explanation forthe increased tenderness after EE treatment (Table1). In controls, cathepsin B + L activity in themyofibrillar and soluble fraction were greater than inEE-treated samples (Table 3), and cathepsin B and Lare known to have optimum activity at pH 5.5.Therefore, the cathepsins could have contributed tothe overall net myofibrillar proteolysis in porcine LMat the normal ultimate pH and, thus, resulted indifferences in protein degradation between control andtreated samples.

Implications

Epinephrine and exercise treatment caused ahigher ultimate pH and an increase in meat tender-ness. This treatment increased the proteolytic poten-tial of m-calpain and decreased cathepsin B + Lactivity in a myofibrillar and a soluble fraction. Theincrease in calpain activity may have been due to aneffect of epinephrine resulting in an increased level of

m-calpain in the living animal. Epinephrine andexercise treatment also increased degradation of a39-kDa band and reduced appearance of bands at 31and 24 kDa in postmortem muscle. Degradationproducts at 30, 31, and 32 kDa were labeled bytroponin-T antibody. The 24-kDa band was identifiedas a troponin-I degradation product. The tenderizationmechanism of the epinephrine and exercise treatmentis likely to involve pH-induced swelling due toincreased water-holding capacity and m-calpain-in-duced degradation of myofibrillar proteins.

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