Elevated rate of collagen solubilization and postmortem degradation in muscles of lambs with high growth rates: possible relationship with activity of matrix metalloproteinases.

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.

Coordinate expression of matrix-degrading proteinases and their activators and inhibitors in bovine skeletal muscle.

Matrix metalloproteinases (MMP) responsible for degradation of connective tissue are found in most tissues. The MMP are regulated at the levels of transcription, zymogen activation by plasmin or membrane-type- (MT) MMP, and control of enzyme activity by tissue inhibitors of metalloproteinases (TIMP). Whole bovine skeletal muscle showed multiple MMP activities on gelatin zymography and also expressed mRNA encoding MMP-1, -2, -9, -14, and -16, tissue inhibitors of metalloproteinase (TIMP)-1, -2, and -3 and plasminogen activator and its receptor. Purified intramuscular fibroblasts and myogenic cell culture derived from satellite cells expressed most or all of these elements. Statistical analysis (n = 35) revealed a strong positive correlation among the mRNA levels of several elements of the MMP system, including MMP-2, MMP-14, TIMP-1, -2, and -3 (r = 0.614 to 0.930, P < 0.0001). Our results provide an extensive profile of an extracellular proteolytic cascade involving MMP in skeletal muscle and suggest that 1) the activation cascades of muscle MMP may be initiated by both plasmin and membrane-type MMP; 2) a group of genes involved in the same "arm" of zymogen activation are coexpressed in this tissue; and 3) skeletal muscle cells, in addition to the intramuscular fibroblasts, express an extensive complement of MMP and related proteins.

Potential m-calpain substrates during myoblast fusion.

Many studies have demonstrated that m-calpain was implicated in cell membrane reorganization-related phenomena during fusion via a regulation by calpastatin, the specific Ca2+-dependent proteolytic inhibitor. However, the real biological role of this protease is unclear because many targeted proteins are still unknown. Using different digestion experiments we have demonstrated that desmin, vimentin, talin, and fibronectin represent very good substrates for this proteinase capable of cleaving them in fragments which are immediately degraded by other enzymatic systems. Concerning intermediate filaments, we showed that during the phenomenon of fusion, the amount of desmin was significantly reduced while the concentration of vimentin presented a steady level. On the other hand, we have conducted biological assays on cultured myoblasts supplemented by exogenous factors such as calpain inhibitors or antisense oligonucleotides capable of stimulating or inhibiting m-calpain activity. The effect of such factors on fusion and concomitantly on the targeted substrates was analyzed and quantified. When m-calpain activity and myoblast fusion were prevented by addition of calpain inhibitors entering the cells, the amounts of desmin, talin, and fibronectin were increased, whereas the amount of vimentin was unchanged. Using antisense strategy, similar results were obtained. In addition, when the phenomenon of fusion was enhanced by preventing calpastatin synthesis, the amounts of desmin, talin, and fibronectin were significantly reduced. Taken together, these results support the hypothesis that m-calpain is involved in myoblast fusion by cleaving certain proteins identified here. This cleavage could modify membrane and cytoskeleton organization for the myoblasts to fuse.

Calpastatin-modulation of m-calpain activity is required for myoblast fusion.

Previous studies have demonstrated a role for m-calpain in myoblast fusion. Moreover, the presence, in differentiated cells, of a highly specific endogenous inhibitor of calpain, calpastatin, has led to the hypothesis that a regulation of or a protection against m-calpain activity by calpastatin could also occur during the earlier stages of muscle cell differentiation. In order to verify this hypothesis, we have investigated, in myoblast culture, the appearance of calpastatin-mRNA and its corresponding protein. Our results provide evidence that calpastatin is already present at the earlier stages of myoblast differentiation and that a significant decrease of the levels of calpastatin mRNA and its protein precedes myoblast fusion. In addition, the induction of an artificial decrease in calpastatin level, via an appropriate antisense oligodeoxyribonucleotide methodology, leads to earlier and faster myoblast fusion. Together with previous studies, these results indicate that m-calpain and calpastatin are functionally involved in myoblast fusion. Our findings also demonstrate that an acute "hyperactivity" of m-calpain resulting from the decrease of calpastatin synthesis is necessary during the early stages of this step of differentiation.

Myoblast fusion requires fibronectin degradation by exteriorized m-calpain.

We recently reported that when myoblasts fuse, m-calpain could be exteriorized. Indeed, at present a number of works support this hypothesis because this enzyme was localized intercellularly and more particularly associated to extracellular matrix components. Knowing that the cell surface of the fusing myoblast is supposed to undergo many changes, we addressed the question whether m-calpain could be involved in the phenomenon of fusion via fibronectin cleavage or degradation. Using different digestion experiments, we demonstrated that soluble purified fibronectin and highly insoluble fibronectin fibrils represent very good substrates for this proteinase; moreover, at the burst of fusion, fibronectin proteolytic fragments could be identified. On the other hand, we have conducted biological assays on cultured myoblasts using a defined medium supplemented by exogenous factors capable of stimulating or inhibiting m-calpain activity. The effects of such factors on rat myoblast fusion and concomitantly on the targeted glycoprotein were analyzed and quantified. When m-calpain activity and the phenomenon of fusion were reduced (defined medium without insulin), the amount of the 220-kDa fibronectin band was increased by 43%. When m-calpain activity and myoblast fusion were prevented by addition of antibodies to m-calpain or calpain inhibitor II, the fibronectin concentration was higher since it was increased by approximately 67 and approximately 71%, respectively. In addition, when observed at the ultrastructural level, m-calpain seems to be localized at the potential fusion site of myoblasts and more particularly associated to the extracellular matrix when muscle cells were initially treated by anti-m-calpain IgG. Taken together, these results support the hypothesis that exteriorized m-calpain could be, in part, involved in myoblast fusion via fibronectin alteration or degradation.

Evidence for implication of muscle-specific calpain (p94) in myofibrillar integrity.

The expression and the putative function(s) of a specific muscle calcium-dependent protease were investigated during myogenesis using rat myoblast primary cultures as a model. We have shown that the levels of p94 mRNAs increase as a function of myoblast differentiation, with the greatest amount of these RNAs being present during the later stages (8th day after plating). After an antisense oligodeoxyribonucleotide treatment with p94, ultrastructural studies show dramatic perturbations in differentiated myotubes and during myofibrillogenesis, mainly involving myofibrillar stability and Z-line integrity. These results may be related to recent findings about the role of p94 gene mutations in limbgirdle muscular dystrophy type 2A.

An antisense oligodeoxyribonucleotide to m-calpain mRNA inhibits myoblast fusion.

Previous studies have led to the hypothesis of a possible role for m-calpain (EC 3.4.22.17) in myoblast fusion in culture in vitro. To support this hypothesis, an antisense strategy has been used with cultured primary rat myoblasts. Using an appropriate antisense oligodeoxyribonucleotide to m-calpain mRNA, an inhibition of myoblast fusion has been observed, the maximum being obtained when the cell culture was treated with 30 microM of oligomer. Synthesis of m-calpain was decreased by 48% while high concentrations of antisense oligonucleotide do not significantly affect myoblast proliferation. The specificity of m-calpain intervention during fusion has also been confirmed using antisense oligonucleotides to mu-calpain and p94 mRNAs, respectively.

Desmin degradation and Ca(2+)-dependent proteolysis during myoblast fusion.

It has already been reported that, in vitro, intermediate filaments such as desmin and vimentin are very susceptible to proteolysis by calpains (Ca(2+)-activated cysteine proteinases). On the other hand, desmin and m-calpain are both present at the onset of myoblast fusion and throughout this phenomenon. Based on these observations, the aim of this study was to demonstrate, with cultured rat myoblasts, that the amount of desmin decreased significantly as multinucleated myotubes were formed. Using immunoblot analysis, it has been shown that the desmin concentration decreased 41% as myoblasts fuse. Moreover, under conditions which stimulate myoblast fusion, desmin concentration was reduced by 21% compared to the control culture. Under our experimental conditions, which lead to a reduced desmin level, the amount of m-calpain was increased about three-fold. These results suggested that m-calpain could be involved in myoblast fusion via desmin cleavage. This hypothesis was confirmed by the results obtained after calpeptin treatment. In the presence of this cell-penetrating inhibitor of calpains, desmin seems not to be degraded. Taking into account the observations obtained after different hydrolysis assays and as compared to those observed on cultured cells, it seems conceivable that m-calpain would be able to initiate desmin cleavage leading to the formation of proteolytic fragments which should be immediately degraded.

Rat myoblast fusion requires exteriorized m-calpain activity.

Our previous studies demonstrated that fibronectin could be proteolyzed by m-calpain during muscle cell differentiation. Recent results indicated also that m-calpain could be exteriorized and more particularly associated to extracellular matrix components. To clarify one of the possible physiological functions of this proteinase during myogenesis, we have analyzed the incidence of added purified m-calpain and calpain inhibitors on the fusion kinetics of cultured myoblasts. Our results provided evidence that at low concentration (0.01 microgram/ml), added m-calpain induces precocious fusion and increases myoblast fusion by 78%. At high concentrations (10 micrograms/ml), the viability of the cells was not affected but the myoblasts were unable to fuse. Leupeptin and calpastatin--potent m-calpain inhibitors--added to the culture medium reduced myoblast fusion by 70%. On the other hand, the addition of monospecific m-calpain polyclonal antibodies to the culture medium induced a 76% decrease of myoblast fusion. In order to trap exteriorized m-calpain, myoblasts were incubated for 24 h with m-calpain antibodies. Following this treatment, nonpermeabilized myoblasts exposed to labeled secondary antibodies showed fluorescent spots scattered at the cell surface. These results strongly support that m-calpain which was involved in myoblast fusion was exteriorized and suggest therefore that this enzyme may play an important role extracellularly.

Evidence for fibronectin degradation by calpain II.

Recent work supports the hypothesis that calpain II can be exteriorized. Indeed, this cysteine calcium-dependent proteinase was shown to be intercellularly, and, more particularly, associated to extracellular matrix components. Thereby, calpain II could be involved in hydrolysis of pericellular matrix components such as fibronectin, which is known to play an important role in cellular differentiation. Our in vitro studies provide evidence that fibronectin is a potential substrate for calpain II. On cultured cells, our findings show that calpain II is able, on the one hand, to cleave the fibrillar network of fibronectin secreted by fibroblasts, and, on the other, to decrease dramatically the fibronectin amount secreted by myoblasts just before fusion. Moreover, following this treatment, myoblasts become spherical due to the cleavage of this attachment factor. However, these cells, plated on an appropriate substrate are still able to differentiate. Our results suggest that calpain II is indeed involved in myoblast fusion via the fibronectin cleavage since it is well established that myogenic lineages lose this glycoprotein at the time of fusion.