Proteomic study of calpain interacting proteins during skeletal muscle aging.

Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern. Although sarcopenia is well documented, the molecular mechanisms of this condition still remain unclear. The calcium-dependent proteolytic system is composed of calcium-dependent cysteine proteases named calpains. Calpains are involved in a large number of physiological processes such as muscle growth and differentiation, and pathological conditions such as muscular dystrophies. The aim of this study was to determine the involvement of this proteolytic system in the phenotype associated with sarcopenia by identifying key proteins (substrates or regulators) interacting with calpains during muscle aging. Immunoprecipitations coupled with proteomic analyses and protein identification by mass spectrometry have been undertaken. Reverse co-immunoprecipitation, cellular colocalisation by confocal microscopy and calpain-dependent in vitro proteolysis of several of the identified proteins have been also carried out. We identified ATP synthase subunit alpha and alpha actinin 3 as key partners of calpains during muscle aging. Such interactions would suggest that calpains are implicated in many processes altered during aging including cytoskeletal disorganisation and mitochondrial dysfunction.

Calcium-dependent proteolytic system and muscle dysfunctions: a possible role of calpains in sarcopenia.

The calcium-dependent proteolytic system is composed of cysteine proteases named calpains. They are ubiquitous or tissue-specific enzymes. The two best characterised isoforms are the ubiquitously expressed mu- and m-calpains. Besides its regulation by calcium, calpain activity is tightly controlled by calpastatin, the specific endogenous inhibitor, binding to phospholipids, autoproteolysis and phosphorylation. Calpains are responsible for limited proteolytic events. Among the multitude of substrates identified so far are cytoskeletal and membrane proteins, enzymes and transcription factors. Calpain activity is involved in a large number of physiological and pathological processes. In this review, we will particularly focus on the implication of the calcium-dependent proteolytic system in relation to muscle physiology. Because of their ability to remodel cytoskeletal anchorage complexes, calpains play a major role in the regulation of cell adhesion, migration and fusion, three key steps of myogenesis. Calcium-dependent proteolysis is also involved in the control of cell cycle. In muscle tissue, in particular, calpains intervene in the regeneration process. Another important class of calpain substrates belongs to apoptosis regulating factors. The proteases may thus play a role in muscle cell death, and as a consequence in muscle atrophy. The relationships between calcium-dependent proteolysis and muscle dysfunctions are being further developed in this review with a particular emphasis on sarcopenia.

Myoblast migration is prevented by a calpain-dependent accumulation of MARCKS.

Calpains, also called calcium activated neutral cysteine proteases are presently known to play pivotal roles in physiological and biological phenomena such as signal transduction, cell spreading and motility, apoptosis, regulation of cell cycle and regulation of muscle cell differentiation. Concerning this last point, calpains have been shown to play a crucial role during the earlier myogenesis. In this study we have analyzed the involvement of calpains during an important step of myogenesis: myoblast migration. Our findings show that myoblast migration was drastically reduced when the expression of micro- and m-calpain was decreased. We have also observed that MARCKS (myristoylated alanine rich C kinase substrate), a protein localized at focal adhesion sites, was significantly accumulated when the expression levels of calpains were decreased. Also, using phorbol myristate acetate, (an activator of PKC) and plasmids carrying the full-length cDNA of MARCKS or a cDNA fragment lacking the phosphorylation site domain, we demonstrated that normal myoblast migration is dependent on MARCKS phosphorylation and localization.

Protein kinase Calpha is a calpain target in cultured embryonic muscle cells.

Previously we isolated a micro-calpain/PKCalpha complex from skeletal muscle which suggested tight interactions between the Ca2+-dependent protease and the kinase in this tissue. Our previous studies also underlined the involvement of ubiquitous calpains in muscular fusion and differentiation. In order to precise the relationships between PKCalpha and ubiquitous calpains in muscle cells, the expression of these two enzymes was first examined during myogenesis of embryonic myoblasts in culture. Our results show that calpains and PKCalpha are both present in myotubes and essentially localized in the cytosolic compartment. Moreover, calpains were mainly present after 40 h of cell differentiation concomitantly with a depletion of PKCalpha content in the particulate fraction and the appearance of PKMalpha fragment. These results suggest a possible calpain dependent down-regulation process of PKCalpha in our model at the time of intense fusion. In our experimental conditions phorbol myristate acetate (PMA) induced a rapid depletion of PKCalpha in the cytosolic fraction and its translocation toward the particulate fraction. Long term exposure of myotubes in the presence of PMA induced down-regulation of PKCalpha, this process being partially blocked by calpain inhibitors (CS peptide and inhibitor II) and antisense oligonucleotides for the two major ubiquitous calpain isoforms (m- and micro-calpains). Taken together, our findings argue for an involvement of calpains in the differentiation of embryonic myoblasts by limited proteolytic cleavage of PKCalpha.

Evidence for a MARCKS-PKCalpha complex in skeletal muscle.

MARCKS (Myristoylated Alanine Rich C Kinase Substrate) is a protein known to cross-link actin filament and consequently, is very important in the stabilization of the cytoskeletal structure. In addition, it has been recently demonstrated that the phosphorylation rate of this protein changes during myogenesis and that this protein is implicated in fusion events. For a better understanding of the biological function of MARCKS during myogenesis, we have undertaken to identify and purify this protein from rabbit skeletal muscle. Three chromatographic steps including an affinity calmodulin-agarose column were performed. The existence of a complex between the two proteins was confirmed by non-denaturing gel electrophoresis and immunoprecipitation. Two complexes were isolated which present an apparent molecular weight of about 600 kDa. Such interactions suggest that MARCKS is either a very good PKCalpha substrate and/or a regulator of PKC activity. These results are supported by previous studies showing preferential interactions and co-localization of PKC isozyme and MARCKS at focal adhesion sites. This is the first time that MARCKS has been purified from skeletal muscle and our data are consistent with a major role of this actin- and calmodulin-binding protein in cytoskeletal rearrangement or other functions mediated by PKalpha. Our results provide evidence for a tight and specific association of MARCKS and PKCalpha (a major conventional PKC isozyme in skeletal muscle) as indicated by the co-purification of the two proteins.

Calpain-PKC inter-relations in mouse hippocampus: a biochemical approach.

In previous studies, we isolated and identified a mu-calpain/PKCalpha complex from rabbit skeletal muscle. Here, we have used specific purification procedures in order to study the interactions between mu-calpain and PKC in mouse hippocampus, a brain structure implicated in memory processes. We observed that mu-calpain and conventional PKCs (alpha, betaII and gamma) are co-eluted after anion exchange chromatography. In contrast to our previous results obtained on skeletal muscle, mu-calpain and PKC isoenzymes were dissociated after gel filtration chromatography. Furthermore, mu-calpain induced the proteolytic conversion of PKCalpha, betaII, and gamma into PKMalpha, betaII, and gamma with a preferential hydrolysis of PKCgamma, a specific isoenzyme of the nervous system. Although the mu-calpain/PKC interactions in the hippocampus are quite different from skeletal muscle, our results however, point out the functional importance of these inter-relations. Moreover, as PKCgamma has been involved in the biochemical events underlying learning and memory, the preferential relationship between mu-calpain and PKCgamma promotes the importance of the role that mu-calpain could play in the cellular mechanisms of memory formation.

Degradation of protein kinase Malpha by mu-calpain in a mu-calpain-protein kinase Calpha complex.

In previous studies, we isolated and identified a mu-calpain-PKCalpha complex from rabbit skeletal muscle. At the same time we pointed out that an association between mu-calpain and PKCalpha could occur at the level of the plasma membrane of muscle cells, and that PKCalpha could thus be considered as a potential mu-calpain substrate. In the present study, using the mu-calpain-PKCalpha complex as a model, we report that mu-calpain is activated in the combined presence of physiological calcium concentrations (less than 1 microM) and phosphatidylserine. Furthermore our data also show that: (1) there exists a correlation between the appearance of autolyzed mu-calpain forms and PKCalpha hydrolysis which leads to the formation of PKMalpha; (2) in certain experimental conditions, autolyzed mu-calpain forms are able to hydrolyze PKMalpha independently of the presence of diacylglycerol.

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.

Ca(2+)-dependent proteinases (calpains) and muscle cell differentiation.

The chronology of appearance of calpain I and calpain II was analyzed during myogenesis of embryonic myoblasts in culture. The influence of the hormones insulin and corticosterone, and insulin growth factor-1 (IGF-1) and transforming growth factor-beta (TGF-beta) on the modulation of calpain-calpastatin levels during myogenesis was also analyzed. Immunodetection assays using specific antibodies and enzymic activities showed that during muscle cell differentiation in vitro, calpain II is present from the beginning of myoblast fusion (2nd day) increasing until the 6th day and then reaching a plateau. These observations were confirmed by an analysis of the expression of total calpain mRNAs which followed the same time profile, thereby providing evidence for a transcriptional regulation in the expression of calpains. Even if an increase in calpain II activity occurs at approximately the same time as an increase of fusion, calpain II activity and rate of fusion are not closely correlated. The involvement of calpain II in some event that follows myoblast fusion is suggested. On the other hand, calpain I and calpastatin were detected only on the 6th day of cell culture growth; these results enable us to argue that if calpain I has any biological role (which remains to be established), this role occurs during the final stages of muscle cell differentiation. The presence of exogenous factors which are known to affect muscle cell differentiation by altering either the rate of protein synthesis, or degradation or both, significantly affects the modulation of calpain-calpastatin levels. Such a regulation at the transcriptional level suggests that calpains do not act as housekeeping enzymes during myogenesis.

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.

Quantitative measurement of calpain I and II mRNAs in differentiating rat muscle cells using a competitive polymerase chain reaction method.

Levels of calpain I and calpain II mRNAs were analyzed at different stages of rat skeletal myoblast differentiation using a competitive polymerase chain reaction method. The results provide evidence that only calpain II mRNAs were present in significant quantities on the second day while calpain I mRNAs were identified on the fourth day of differentiation. If there is no compelling reason to believe that synthesis of calpains I and II is regulated at the level of mRNA, our results suggest that calpain II will be more particularly involved in Ca(2+)-mediated events accompanying myoblast fusion. On the other hand, calpain I, because of its later appearance may probably act on specific substrates such as myofibrillar proteins, associated myofibrillar proteins or the control of enzyme metabolism. Added factors such as insulin, which is known to induce enhancement of myoblast growth or myoblast fusion, had a significant effect on the amounts of calpain I and II mRNAs. In the presence of TGF-beta, a potent inhibitor of myoblast fusion, calpain I and II mRNAs were decreased. These results confirm first that a Ca(2+)-dependent proteolytic system is positively correlated with myoblast fusion (via calpain II) and second, that transcriptional regulation of calpains I and II may be negatively modulated during myoblast differentiation.

In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II.

Dystrophin is a cytoskeletal protein which is thought to play an important role in membrane physiology since its absence (due to gene deficiency) leads to the symptoms of Duchenne muscular dystrophy (DMD). Some disruption in the regulation of intracellular free Ca2+ levels could lead to DMD-like symptoms. In this study, calpains, which are very active calcium-dependent proteases, were examined for their capacity to hydrolyse dystrophin in vitro. The results show that calpains are able to split dystrophin and produce breakdown products of different sizes (the degree of cleavage being dependent on the incubation time with proteases). The time-course of protease degradation was examined by Western immunoblot using three polyclonal sera which were characterized as being specific to the central (residues 1173-1728) and two distal parts of the molecule ie specific to the N-terminal (residues 43-760) or the C-terminal (residues 3357-3660) extremities of the dystrophin molecule. The cleavage patterns of dystrophin showed an accumulation of some major protease-resistant fragments of high relative molecular mass (250-370 kDa). These observations demonstrate that calpains digest dystrophin very rapidly when the calcium concentration is compatible with their activation. For instance, it is clear that calpains first give rise to large dystrophin products in which the C-terminal region is lacking. These observations suggest that dystrophin antibodies specific to the central domain of the molecule should be used to detect dystrophin for diagnostic purposes and before any conclusion as to the presence or absence of dystrophin can be deduced from results obtained using immunoanalyses of muscle biopsies.