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Elevated rate of collagen solubilization and postmortem degradation inmuscles of lambs with high growth rates: Possible relationship with activity of matrix metalloproteinases

M. N. Sylvestre^*, D. Balcerzak, C. Feidt^*, V. E. Baracos and J. Brun Bellut^*,1

^* Laboratoire de Sciences Animales, INRA-INPL-UHP, 54505 Vandoeuvre-lès-Nancy, France and and Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5

¹ Correspondence:
Nancy I, ENSAIA, BP 172 (phone: 33 (0)-3-83-59-58-89; fax: 33 (0)-3-83-59-58-89; E-mail:
brunbell{at}ensaia.inpl-nancy.fr).

	Abstract

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The extracellular matrix, composed mainly of collagen, is considered responsible for the residual toughness of meat. Matrix metalloproteinases (MMP) responsible for the degradation of connective tissue are found in most tissues, but their participation in meat aging has not been tested. We recently showed that skeletal muscle has multiple MMP activities, as well as regulators and tissue inhibitors of metalloproteinases. Here we present the first observations of physiologic and postmortem variation of MMP activities in muscle. Growing lambs were offered two levels of intake: hay + concentrate for lambs with high growth rate (average daily gain > 250 g) and hay only for those with low growth rate (average daily gain < 25 g). At slaughter and at 21 d of postmortem aging of longissimus and semimembranosus muscles, we studied collagen content, collagen solubility, free hydroxyproline (OH-pro), and levels of latent and active forms of a matrix metalloproteinase (MMP-2) by gelatin zymography. Our results demonstrate the presence of an active isoform of MMP-2 in lamb muscle. Its level was higher (+90%, P < 0.01) in lambs that expressed a high growth rate. Activity of MMP-2 was also present at 21 d postmortem, at levels similar to those detected at slaughter. At slaughter and at 21 d, all muscles contained latent MMP-2 and the quantity of proenzyme was greater than that present in the activated form. The levels of free OH-pro in muscles of lambs with high growth rate increased significantly (P < 0.001) over 21 d from 3.75 to 5.08% of total collagen, and this was significantly related to the level of active MMP-2 at slaughter. By contrast, the amount of free OH-pro in muscles of lambs with low growth rate was not different at 21 d (1.63% of total OH-pro) than it had been at slaughter (1.84% of total OH-pro). These results suggest that collagen degradation all the way to free amino acids occurs postmortem in muscle and that there are active MMP simultaneously present that may account for this catabolism. The growth rate of animals at slaughter influences collagen turnover in vivo, as well as postmortem collagen degradation.

Key Words: Aging • Collagen • Growth • Hydroxyproline • Lambs • Metalloproteinase

	Introduction

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The structural integrity of collagen has been considered to be unaffected during postmortem aging of meat (Bailey, 1985). However, changes in its mechanical (Nishimura et al., 1998), physicochemical (Judge and Aberle, 1982; Stanton and Light, 1990), and ultrastructural (Liu et al., 1995; Nishimura et al., 1995) properties have been observed. The collagen properties have been studied in relation to animal factors and production systems (Miller et al., 1987; Boutten et al., 2000), but little is known about its postmortem catabolism (Aberle et al., 1981; Fishell et al., 1985) and there are no published data about physiologic and postmortem variation in the proteolytic system that degrades collagen in any species.

The matrix metalloproteinase (MMP) system, including collagenases, stromelysins, and gelatinases, degrades connective tissue proteins in all tissues. Collagen denatured by collagenases can be degraded to small peptides by gelatinase activity (MMP-2 or MMP-9) (Seltzer et al., 1981, 1990). The final step in collagen degradation is the release of hydroxyproline (OH-pro), which is used as a specific indicator of this catabolism (Kivirikko, 1970). Collagen turnover and breakdown in vivo are clearly influenced by physiological factors. The appearance of OH-pro in plasma and urine is elevated in animals expressing a high growth rate (Bruce et al., 1991). Enzymes of the MMP system, like other muscle proteases, might express activity postmortem. The products of this activity (free OH-pro and peptides containing OH-pro) in meat aging have been very little studied. Feidt et al. (1996) showed an appearance of free OH-pro during beef aging.

The aim of the study was to determine the effect of growth rate (high vs slow) on the level of pro- and MMP-2 in muscles and collagen alteration indicators (collagen solubility and OH-Pro).

	Materials and Methods

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Experimental Design and Feeding
A total of 18 lambs (INRA 401-Charolais breed) were used in two experiments. Growth rate variation was induced by feeding two levels of intake: hay + concentrate in the case of lambs expressing a high growth rate and hay only for lambs with a low growth rate. In Exp. 1, male lambs were studied (n = 3/diet treatment). Lambs were 97.5 (± 14.7) d old and weighed 27.1 (± 1.2) kg at the beginning of the experiment. Animals were housed in individual pens during an experimental period of 21 d. In Exp. 2, both male and female lambs were studied (n = 3 of each sex/diet treatment). Lambs were 77 (± 5.7) d old and weighed 19.6 (± 2.2) kg at the beginning of the experiment. Animals were housed in individual pens during an experimental period of 26 d.

All animals were provided hay for ad libitum consumption (crude fiber: 28.6; crude protein: 10.6; total ash: 8% of dry matter). The lambs with high growth rate additionally received 900 g/d of a Baron Starter concentrate (crude fiber: 8; crude protein: 19.4, and total ash: 9.2% of dry matter) twice a day. The animals were weighed weekly (before the evening meal) and before slaughter.

Slaughter and Muscle Sampling
Lamb carcasses were trimmed, transferred into a cold room (4°C) 1.5 h after slaughter, and stored for 21 d. Within 10 min of the time each animal was killed, one sample of 50 to 100 g from each longissimus muscle in Exp. 1 and 2 and semimembranosus muscle in Exp. 1 was taken for analysis. An additional sample (30 to 50 g) of these two muscles was taken and immediately frozen in liquid nitrogen for enzyme (MMP) detection by zymography. Twenty-four hours after slaughter, whole longissimus and semimembranosus muscles were excised from carcasses and divided into two portions. Samples were soaked (1 min) in 0.1 g/L of sodium azide (NaN₃, Merck, Fontenay-sous-bois, France) to prevent bacterial growth, vacuum-packed, and stored at 4°C up to 21 d postmortem. At d 21, one sample was frozen at -80°C for determination of MMP activities. At the time of analysis, the parts of the muscles in contact with NaN₃ were trimmed and the samples were dissected to remove visible connective tissue.

Analysis
Total Collagen Content.
The total collagen content was estimated from the OH-pro content of 3 g of minced muscle using the method of Bonnet and Kopp (1986). Collagen content was expressed as micrograms of OH-pro per gram of fresh muscle.

Collagen Insolubility.
Collagen insolubility was estimated for each sample in triplicate. A solution of Trizma hydrochloride (Tris-HCl: 0.02 M, pH 7.4; Sigma Chemical Co., Chesnes, France) containing 0.23 M NaCl was added to minced fresh muscle (5 mL/g). The suspension was mechanically agitated, incubated 15 min at room temperature, heated to 90°C for 3 h in a temperature-controlled water bath, then filtered on Whatman paper (#113). Insoluble collagen (IC) was retained on the filter but soluble collagen (SC) was found in the filtrate. Filters (with IC) were oven-dried (100°C for 24 h) then incubated 4 h at 100°C in 15 mL of 70% perchloric acid (HCLO₄, Fisher, Elancourt, France) to hydrolyze the collagen. The filtrate, containing SC, was freeze-dried (48 h) and then incubated as described above to hydrolyze the collagen. The OH-pro content of the SC and IC fractions was estimated by the method described by Bonnet and Kopp (1986). Collagen insolubility, in each aliquot, was expressed by the ratio IC/(IC + SC). The precision of the method expressed from the coefficient of variation obtained on the triplicates was 3% for Exp. 1 and 8% for Exp. 2.

Free OH-pro of the Nonprotein Nitrogen Fraction.
The nonprotein nitrogen (NPN) fraction of muscle was prepared according to Sylvestre et al. (2001). Twelve grams of muscle was homogenized with an Ultra-Turax in 3.26% HClO₄ and centrifuged for 20 min at 4,000 x g (4°C). The supernate was neutralized with 2 M potassium carbonate (Prolabo, Fontenay-sous-bois, France), filtered, centrifuged for 20 min at 12,000 x g (4°C), and then frozen at -18°C until assayed. Free OH-pro was determined by HPLC after phenyl-isothiocyanate derivatization (Sigma Chemical Co., Chesnes, France) with norleucine as internal standard. Tissue free OH-pro contents were expressed as micrograms of OH-pro per gram of fresh muscle.

Detection of MMP Activities: Gelatin Zymography.
Sample preparation, electrophoresis, and MMP zymography were done essentially as described by Balcerzak et al. (2001).

Sample Preparation.
After grinding in liquid nitrogen, tissue samples were incubated (24 h at 4°C) in extraction buffer (0.01 M cacodylic acid, pH 5.2, 0.15 M calcium chloride (CaCl₂), 0.15 mM zinc chloride, 2 mM NaCl) (1 mL buffer/100 mg tissue). Following the incubation, the sample was homogenized with a Polytron (Brinkman, Mississauga, ON, Canada) and centrifuged for 10 min at 350 x g at 4°C. The pellet was then resuspended in extraction buffer (1 mL buffer/100 mg tissue), rehomogenized, and centrifuged. This step was repeated twice and all of the supernates were pooled. Soluble proteins were quantified by the BCA protein assay (Pierce, Rockford, IL) using BSA as a protein standard.

Electrophoresis and Gelatin Zymography.
Samples were mixed with 0.25 volumes of nonreducing sample buffer consisting of 0.3 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 0.03% bromophenol blue. Electrophoresis was run on 15% SDS-PAGE gels containing gelatin (Type I, 1 mg/mL [Sigma Chemical Co.]). After electrophoresis, the gel was removed and incubated for 30 min at 25°C in Triton X-100 (2.5% in distilled water). After two 15-min washes in Tris-HCl (50 mM, pH 7.5), gels were incubated 20 h at 37°C, with gentle shaking, in 50 mM Tris-HCl, pH 7.5, 10 mM CaCl₂, 0.05% Brij-35. Gels were stained with 0.1% Naphthol blue-black solution in acetic acid/methanol/distilled water (1/4.5/4.5, vol/vol/vol) and destained with distilled water.

Semiquantitative Zymography.
In the presence of gelatinase activity, gelatin incorporated into the gel is degraded, leaving clear bands against a stained background after gel staining. The intensity of these bands is directly proportional to the quantity of gelatin degraded, which is correlated with the quantity of enzyme present in the sample (Kleiner and Stetler-Stevenson, 1994). Both the proenzyme and the active form show a signal in this assay and picogram quantities can be detected (Kleiner and Stetler-Stevenson, 1994). In preliminary experiments the intensity of the bands appearing in the zymograms was determined as a function of the quantity of total protein loaded on the gel. The linear portion of this relationship was identified and the quantity of total protein from the middle of this linear range (15 µg) was selected for comparative analysis of samples from different animals. In studies of lambs with different growth rates, we ran each sample three times (i.e., on three separate gels) and the variation on the intensity of the band between the different runs for the same sample was less than 3%.

Statistical Analysis
Results were analyzed using two-way analysis of variance (growth rate and muscle in the first experiment, growth rate and sex in the second). The means were compared by Newman-Keuls test (STAT-ITCF, Boigneville, France). The effect of postmortem aging (d 0 against d 21) was tested by analysis of variance. This model included the fixed effects of the treatment (growth, muscles, and[or] sex), time of aging, and the interaction time of aging x treatment.

	Results

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In both experiments, lambs fed with hay and concentrate grew (P < 0.01) faster than those that received hay alone (Table 1). No significant difference in growth rate was observed between male and female lambs in Exp. 2 (data not shown).

Muscles of all lambs studied showed MMP activity on gelatin zymography. One major gelatinase activity was observed (Figure 1) in the homogenate of longissimus and semimembranosus muscles. This activity had an apparent molecular weight of 66 kDa (the major signal) characteristic Pro-MMP-2. A less abundant activity with an apparent molecular weight of 62 kDa corresponds to the activated form of MMP-2. No gelatinase activity with molecular weight corresponding to MMP-9 or its proenzyme was detected in these samples. It appears that the majority ( 95.3%) of total MMP-2 exists in its zymogen form in lamb muscle, as we previously observed for beef (Balcerzak et al., 2001). When these bands were quantified by scanning densitometry, the level of active MMP-2 was higher (+90%, P < 0.01) in the lambs expressing a high growth rate (Table 1); no significant difference (P < 0.204) was observed in the level of Pro-MMP-2 between animals with low and high growth rates (Table 1).

View larger version (30K): [in this window] [in a new window]   Figure 1. Detection of gelatinase activities by zymography in longissimus and semimembranosus muscles at slaughter (d 0) and at 21 d postmortem (Exp. 1). Proteins (15 µg) were separated on a 15% SDS-PAGE gel containing gelatin (1 mg/mL). After migration the gel was washed in a triton X-100 solution (2.5% in distilled water), incubated 20 h at 37°C in enzyme buffer, and stained with Naphthol blue black solution. Lanes from left to right contained the animal number corresponding to high (numbers: 1, 3, and 6) and low (numbers: 2, 4 and 5) growth rate. MMP-2, matrix metalloproteinase type 2 (active form); Pro-MMP-2, zymogen form of MMP-2.

Effect of Growth Rate on Collagen Content and Matrix Metalloproteinase Activity at 21 d of Postmortem Maturation
Experiment 1.
The total collagen fraction became more soluble (by 13.8%, P < 0.001) over 21 d of maturation in all animals studied, regardless of muscle type. After 21 d of aging, collagen of the lambs expressing a high growth rate remained (P < 0.01) less insoluble than that of lambs with low growth rate (68.9% vs 78.5%; Table 1). During the 21 postmortem days, the muscle levels of free OH-pro increased (by 39.3%, P = 0.016) and there was an interaction (P = 0.017) between growth rate and changes in free OH-pro during meat aging. The levels of free OH-pro in lambs with high growth rate increased (P = 0.0142) over 21 d from 3.11 to 5.08% of total collagen (Table 1). By contrast, the amount of free OH-pro in muscles of lambs with low growth rate was not different at 21 d (1.63% of total OH-pro) from that at slaughter (1.61% of total OH-pro).

Muscles of all lambs studied continued to show MMP activity on gelatin zymography at 21 d of postmortem maturation (Figure 1). The levels of active MMP-2 and Pro-MMP-2 did not differ significantly between the two muscles, and no significant interaction was observed between growth rate and muscle type (data not shown). Active MMP-2 levels did not change (P = 0.266) during aging (d 0: 2.67 and d 21: 3.10 arbitrary unit/mg of tissue). The difference observed between the two groups at slaughter for active MMP-2 was still evident at d 21 (Table 1).

The levels of Pro-MMP-2 activity decreased (-32.6% overall, P < 0.0002) during the 21 d of storage. This decrease was -38.9% in the case of lambs with high growth rate and -25.3% for the lambs with low growth rate; by d 21, no significant difference was observed between the lambs expressing high and low growth rates (Table 1).

We observed correlations (P < 0.05) between the levels of active MMP-2 at slaughter and the increase in free OH-pro over the next 21 d (r = 0.614), the level of free OH-pro at d 21 (r = 0.589), as well as the increase in the fraction of free/total OH-pro (r = 0.668) over the next 21 d. In addition, the level of active MMP-2 at slaughter was correlated (P < 0.05) with collagen insolubility at slaughter (r = -0.778) and at d 21 postmortem (r = -0.632). By contrast, we did not observe a significant relationship between the level of active MMP-2 at time of slaughter and the change in collagen solubility over the studied period (Table 2).

Male lambs tended (P = 0.094) to have more free OH-pro than females (15.2 µg OH-pro/g fresh muscle vs 10.9 µg OH-pro/g fresh muscle), but the percentage of this as a fraction of total OH-pro did not differ significantly (3.36 vs 2.36%, P = 0.105).

As observed in the first experiment, muscles of all lambs studied continued to show MMP activity on gelatin zymography at 21 d of postmortem maturation (data not shown). Male and female lambs presented the same levels of active MMP-2 and Pro-MMP-2 and no significant interaction was observed between growth rate and sex (data not shown). There continued to be MMP-2 and Pro-MMP-2 activities at 21 d; however, these were more variable than at the same time point in the first study as well as more variable than they had been in the same animals at slaughter. There were no treatment differences at d 21 in this study in MMP-2 or Pro-MMP-2 activities (Table 1). We observed a strong variability linked to the level of MMP-2 activity of lambs with low growth rate at d 21 (values ranged between 2.28 and 14.16 arbitrary unit/mg fresh muscle). This could explain that at d 21 postmortem there was no significant difference between growth rates.

The correlations observed in Exp. 2 (not shown) were highly consistent overall with those in the first study. We observed a relationship (P < 0.05) between the levels of active MMP-2 at the time of slaughter and the subsequent increase in free OH-pro over the next 21 d (r = 0.777). The level of free OH-pro at d 21 and the increase in the ratio of free/total OH-pro from slaughter to d 21 postmortem were also (P < 0.05) correlated with the level of active MMP-2 at slaughter (r = 0.705 and r = 0.826, respectively). The level of active MMP-2 at slaughter was correlated (P < 0.05) with collagen insolubility at d 21 postmortem (r = -0.692). However, there was no strong relation between the level of active MMP-2 at slaughter and collagen solubilization over the studied period (r = -0.578).

	Discussion

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We modified animal growth by plane of feeding in a manner that would be expected to modify the deposition and turnover rate of tissue proteins. We present the first observations of physiologic and postmortem variation of matrix metalloproteinase activities in skeletal muscle.

Our results provide evidence for the presence of an active isoform of a matrix metalloproteinase (MMP-2) in lamb muscle, and this activity was greater in animals expressing a high growth rate at the time of slaughter. MMP-2 activity was also present at 21 d of post-mortem aging, at similar levels as had been detected at slaughter. Muscles of lambs expressing high growth rates also showed an elevated rate of production of free OH-pro throughout 21 d of meat maturation, and this was significantly correlated with MMP-2 activity. Overall, these observations suggest that matrix metalloproteinases may retain activity for a long time after an animal’s death, and that there is also additional latent MMP activity present for 21 d. Collagen becomes progressively more soluble during postmortem maturation of meat and at least some collagen is completely degraded to free amino acids.

It is unknown to what extent the phenomena of collagen solubilization and collagen degradation are attributable to MMP, with or without the participation of other proteinases. Physiologically, connective tissue catabolism requires the concerted action of collagenases, gelatinases, and stromelysins; each of these enzymes have specificity for unique substrates. In skeletal muscle, the activity and gene expression of MMP 2 are clearly the most abundant; the activity of some of the other enzymes necessary for complete degradation of collagen isoforms as well as activation of MMP is sufficiently low to make detection a considerable challenge (Balcerzak et al., 2001). It is furthermore not clear which of the many elements of the MMP system aside from MMP 2 retain activity postmortem. After death, proteinases such as lysosomal cathepsins can gain access to connective tissue proteins and participate with MMP in a concerted series of autolytic events. Because these enzyme activities are also generally low in muscle, an analysis of amino acid sequence around cleavage sites in key proteins may help to identify which enzymes are operative under postmortem conditions.

Effects of Growth Rate at Slaughter
Several aspects of the intramuscular connective tissue were different between the two treatment groups, including the amount of total collagen per gram of muscle, as well as the fraction of that collagen that was insoluble, soluble, and present as free OH-pro. The total collagen content (per gram of muscle) was lower in the group with high growth rate (by 18 to 33%), consistent with results obtained by others. Aberle et al. (1981), Fishell et al. (1985), and Boccard and Bordes (1986) reported that young cattle with slow growth rate (feeding restriction of energy and[or] protein) presented higher collagen contents in their muscles. This could be a consequence of the relatively advanced growth; several investigations have shown that collagen content relative to myofibrillar protein content falls during normal growth (Boccard et al., 1979; McCormick, 1994).

High growth rate was accompanied by significantly lower insoluble collagen contents (by 29 to 39%). This phenomenon is thought to be attributable to an increased rate of collagen synthesis, with more "neo-synthesised" collagen, which was less polymerized and therefore more soluble. Aberle et al. (1981) and Rucklidge et al. (1992) suggested that elevated growth rate could lead to an increase in the quantity of immature collagen.

Collagen solubility could also be related to different relative rates of proteolysis and to the generation of soluble proteolytic fragments (peptides) and free amino acids. Collagen breakdown on a whole-body basis (determined as urinary OH-pro excretion) is associated with higher growth rates (Kivirikko, 1970). Collagen catabolism rates in muscle have been little studied, but several types of data indirectly suggest that this process is elevated in muscles of animals expressing high growth rates. In our study, these animals had a higher fraction of total collagen present as free OH-pro, the ultimate product of collagen catabolism, as well as higher levels of active MMP-2. Different authors showed that plasma OH-pro levels were higher in animals with fast growth, fed with a high-energy and(or) -protein diet (Wu et al., 1981; Bailey, 1985; Fishell et al., 1985; Bruce et al., 1991). Finally, intracellular degradation of newly synthesised collagen has been described (Bienkowski, 1978). Bienkowski (1984) used a model system of human fibroblasts in culture to show that approximately 15% of the collagen newly synthesized was broken down rapidly during a process termed basal degradation, which is random, continuous, and independent of collagen synthesis. Collagen molecules that enter this pathway (probability of being degraded: 1/6) are not distinguishable from molecules that escape breakdown. This basal mechanism of degradation seemed to be located in endoplasmic reticulum or Golgi apparatus, and enzymes capable of attacking collagen or collagen-derived peptides are located in one of these organelles.

Evolution of Collagen Properties and MMP Activity During Postmortem Aging
During postmortem aging, collagen insolubility decreased in the two groups of lambs (by 12 to 14%), and this change was quantitatively similar to previously published results (Judge and Aberle, 1982; Stanton and Light, 1987; Mills et al., 1989a,b; Stanton and Light, 1990; Liu et al., 1995). Nishimura et al. (1995, 1996a,b, 1998) showed ultrastructural changes in the endo- and perimysium of semitendinosus bovine muscle (appearance of vacuole with various size), clearly visible from 14 to 28 d postmortem. Similarly, Kruggel and Field (1971), using density gradients, and Pfeiffer et al. (1972), by electrophoresis, reported molecular changes in intramuscular collagen of longissimus muscles during an aging period of 21 d at 2°C: cross-link content decreased and breaks appeared between the "polypeptidic" chains of collagen.

After 21 d of storage, collagen from lambs with high growth rate continued to be more soluble than that from more slowly growing lambs. These data confirm those of Aberle et al. (1981) and Fishell et al. (1985). In parallel with increasing solubility of collagen, muscle free OH-pro levels rose during postmortem maturation, and this was related to growth rate. At d 21, the level of free OH-pro was higher in muscles of lambs expressing a high growth rate than it had been at slaughter (+170%; P < 0.001). The fraction of free/total OH-pro was also increased relative to slaughter (4.6% vs 1.1%; P < 0.01; Table 1) in this group of animals. By contrast, the amount of free OH-pro in muscles of lambs with low growth rate was not different at 21 d than it had been at slaughter.

There were several differences between lambs expressing high and low growth rate in muscle collagen, of quite different magnitudes. There were large initial differences in collagen content (18 to 33%) and in the solubility of that collagen (29 to 39%). These differences, attributable to growth rate prior to slaughter, could have an important impact on meat quality. There were also differences in the rate of collagen degradation, based on rates of production of free OH-pro. This degradation, however, corresponded to between 1 and 2% of total collagen for lambs with high growth in our study. At the levels observed here, this phenomenon has a relatively small impact, compared to other factors, on physical properties of the connective tissue in aged meat. However, collagen catabolism could potentially be quantitatively important. If proteolytic activity could be increased, by increasing active MMP at slaughter or by increasing postmortem conversion of latent MMP into their active forms, it seems possible that the fraction of the intramuscular connective tissue that is degraded could be increased.

A number of biochemical questions remain to be clarified. This initial work shows variation in MMP2 activity in muscle that may relate to postmortem collagen catabolism. Numerous elements of the MMP system are known to exist in skeletal muscle and it would be interesting to study its activity at all of its potential control points such as gene expression, zymogen activation (by MT-MMP or by the uPA cascade), and TIMP expression and activity. We have provided evidence for postmortem collagenolysis and MMP-2 activity, but it is not known whether these are causally related. These factors were statistically correlated, such that MMP-2 activity at slaughter was related to the subsequent rate of appearance of free OH-pro postmortem, but this is only suggestive. Interestingly, MMP-2 activity was not related with the change in collagen solubility observed over time postmortem and collagen solubility was not related to OH-pro production rate. Numerous factors are likely to impinge on collagen structure during meat maturation and thus to dictate the presence or absence of various relationships. There are multiple MMP in addition to MMP-2, each with different specificity for collagen types, and it remains to be determined how many of these express activity postmortem. In addition to the proteinases of the MMP system, collagen is susceptible to attack by other hydrolytic enzymes, including lysosomal cathepsins and enzymes that attack intermolecular cross-links (Etherington, 1972; Burleigh et al., 1974; Beltrán et al., 1994, 1997). The complex interplay of these enzymes results in a sequence of fragmentation, progressive solubilization, and eventually complete hydrolysis of connective tissue components. The sequence and sites of attack of the various hydrolases acting postmortem are not known, and it would be important to determine which of these events is limiting for structural change that is related to meat quality.

	Implications

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The majority of scientific research on postmortem proteolysis has concerned the proteinases residing within muscle cells, such as calpains and lysosomal hydrolases, and their actions on myofibrillar proteins. The contribution of matrix metalloproteinases to collagen degradation has been less studied, especially in skeletal muscles of growing animals. Their postmortem activity during meat maturation has never been studied. Our results suggest a small but potentially important contribution of matrix metalloproteinases to the autolytic changes occurring in meat. These results invite further investigation into the antemortem and postmortem regulation of matrix metalloproteinases activity in muscle, including the activation of latent enzyme (zymogen) activity, and the site(s) of attack of collagen isoforms, and their possible relationship to meat quality.

Received for publication August 24, 2001. Accepted for publication February 21, 2002.

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