Calpastatin in rat myoblasts: transient diminution and decreased phosphorylation depend on myogenin-directed myoblast differentiation

The formation of skeletal muscle fibers involves cessation of myoblast division, followed by myoblast differentiation and fusion to multinucleated myofibers. The myogenic regulatory factor myogenin appears at the onset of differentiation; it is required for muscle fiber formation, and cannot be replaced by other factors. The myogenin-dependent pathways and targets are not fully known. Previous studies, indicating an involvement of calpain-calpastatin and caspase in myoblast fusion, were based on the use of various inhibitors. The availability of myogenin deficient cell lines that are incapable of fusion, but regain the ability to differentiate when transfected with myogenin, provide a convenient means to study calpain-calpastatin and caspase in fusing and non-fusing myoblasts without the use of inhibitors. The differentiating wild type myoblasts exhibit decreased calpastatin phosphorylation, transient diminution in calpastatin mRNA, caspase-1 dependent diminution in calpastatin protein, and calpain-promoted proteolysis. In the myogenin-deficient myoblasts, calpastatin phosphorylation is not diminished, caspase-1 is not activated, calpastatin mRNA and protein are not diminished, and protein degradation does not occur. The myogenin-deficient myoblasts transfected with myogenin gene regain the ability to fuse, and exhibit the alterations in calpastatin and proteolysis observed in the wild type cells. Overall, the results demonstrate that the regulation of calpain in these myoblasts is independent of myogenin. In contrast, the regulation of calpastatin depends on myogenin function. The temporary diminution of calpastatin during myogenin-directed differentiation of myoblasts allows calpain activation and calpain-induced protein degradation, required for myoblast differentiation and fusion.     

Caspase-1-induced calpastatin degradation in myoblast differentiation and fusion: cross-talk between the caspase and calpain systems

Previously, we found that calpastatin diminished transiently prior to myoblast fusion (rat L8 myoblasts), allowing calpain-induced protein degradation, required for fusion. Here we show that the transient diminution in calpastatin is due to its degradation by caspase-1. Inhibition of caspase-1 prevents calpastatin diminution and prevents myoblast fusion. Caspase-1 activity is transiently increased during myoblast differentiation. Both calpain and caspase appear to be responsible for the fusion-associated membrane protein degradation. Caspase-1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase-1 in L8 myoblast fusion represents a novel function for this caspase in a non-apoptotic differentiation process, and points to cross-talk between the calpain and caspase systems in some differentiation processes.     

Association of calpain (Ca(2+)-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts.

Calpain isozymes (intracellular, Ca(2+)-dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of mu-calpain and m-calpain (activated by microM and mM Ca(2+), respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti-calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti-calpastatin). Calpastatin was co-precipitated when calpain was immunoprecipitated; calpain was co-precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca(2+)-dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca(2+)levels. It is proposed that the status of cellular calpain-calpastatin association is modulated by cell constituents, for which some possibilities are suggested.     

Biologically active milli-calpain associated with caveolae is involved in a spatially compartmentalised signalling involving protein kinase C alpha and myristoylated alanine-rich C-kinase substrate (MARCKS)

We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.     

Characterization of the calcium-dependent proteolytic system in a mouse muscle cell line

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C₂C₁₂) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.     

Myoblast migration is regulated by calpain through its involvement in cell attachment and cytoskeletal organization

Cell migration is a fundamental cellular function particularly during skeletal muscle development. Ubiquitous calpains are well known to play a pivotal role during muscle differentiation, especially at the onset of fusion. In this study, the possible positive regulation of myoblast migration by calpains, a crucial step required to align myoblasts to permit them to fuse, was investigated. Inhibition of calpain activity by different pharmacological inhibitors argues for the involvement of these proteinases during the migration of myoblasts. Moreover, a clonal cell line that fourfold overexpresses calpastatin, the endogenous inhibitor of calpains, and that exhibits deficient calpain activities was obtained. The results showed that the migratory capacity of C2C12 and fusion into multinucleated myotubes were completely prevented in these clonal cells. Calpastatin-overexpressing myoblasts unable to migrate were characterized by rounded morphology, the loss of membrane extensions, the disorganization of stress fibers and exhibited a major defect in new adhesion formation. Surprisingly, the proteolytic patterns of desmin, talin, vinculin, focal adhesion kinase (FAK) and ezrin, radixin, moesin (ERM) proteins are the same in calpastatin-overexpressing myoblasts as compared to control cells. However, an important accumulation of myristoylated alanine-rich C kinase substrate (MARCKS) was observed in cells showing a reduced calpain activity, suggesting that the proteolysis of this actin-binding protein is calpain-dependent and could be involved in both myoblast adhesion and migration.     

Lack of evidence for the involvement of a β-D-galactosyl-specific lectin in the fusion of chick myoblasts

The role of a β-D-galactosyl-specific lectin, first reported by Teichberg et al., in the fusion of myoblasts in vitro was investigated. The concentration of this lectin in embryonic chick skeletal muscle was found to reach maximal levels at the time of myoblast fusion in vivo. β-D-Galactosyl-β-thiogalactopyranoside and lactose are potent inhibitors of agglutination of trypsinized rabbit erythrocytes caused by the lectin. However, at concentrations of 50 mM these compounds had no effect on either nonsynchronous fusion of myoblasts or on the release of synchronized myoblast cultures from EGTA fusion block. The presence of the agglutinin in the external membranes of chick myoblasts and myotubes could not be demonstrated. It is, therefore, concluded that the involvement of the lectin in the fusion of chick myoblasts remains questionable.     

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.     

Microinjection of calpastatin inhibits fusion in myoblasts

Rat satellite cells (RSC) were microinjected with purified calpastatin or m-calpain, and myoblasts from a C2C12 mouse line were microinjected with purified calpastatin. Microinjection with calpastatin completely prevented fusion of myoblasts from both sources, whereas microinjection with m-calpain significantly increased the rate of fusion of cultured RSC; 44% of the nuclei of RSC cultures were in multinucleated myotubes within 48 h after microinjection with m-calpain plus labeled dextran, whereas only 15% of the nuclei were in multinucleated myotubes after microinjection with dextran alone. Western analyses indicated that neither RSC nor C2C12 myoblasts contained detectable amounts of mu-calpain before fusion. The levels of calpastatin in C2C12 myoblasts increased as cells passed from the proliferative stage to the onset of fusion, and these levels increased substantially in both the C2C12 and the RSC cells as they progressed to the late or postfusion stage. Both RSC and C2C12 myoblasts contained an 80-kDa polypeptide that was labeled with an anti-m-calpain antibody in Western blots. The results are consistent with a role of the calpain system (m-calpain in these myoblast lines) in remodeling of the cytoskeletal/plasma membrane interactions during cell fusion.     

M-calpain levels increase during fusion of myoblasts in the mutant muscular dysgenesis (mdg) mouse

Previous studies have led to the hypothesis of a possible role for the calcium-dependent neutral protease m-calpain in myoblast fusion in culture. To evaluate this hypothesis, we chose as our model, the "muscular dysgenesis" mouse (mdg), which presents in vivo and in vitro characteristics of an elevated process of fusion (Yao and Essien, 1975; Dussartre, 1993; Ashby et al., 1993, Joffroy et al., 1999). The aim of this study was to demonstrate using myoblast cell lines and muscle biopsies from this mdg mutant, that the amount of m-calpain increases significantly as multinucleated myotubes are formed. Using immunoblot analysis, it was shown that the m-calpain concentration in a dysgenic cell line (GLT) increased 3-fold compared to what it was upon the introduction of the differentiation medium. On the other hand, in a normal cell line (NLT), the concentration of m-calpain did not vary significantly. Thus, when the transition from myoblasts to myotubes was slow, and the absolute level of fusion was reduced, as in the NLT cell line, the level of m-calpain was stable. In contrast, when the process of fusion was precocious and fast, and the level of fusion was elevated, such as in the GLT cell line, the concentration of m-calpain increased during fusion. Moreover, when myoblast fusion was prevented by the addition of calpain inhibitor II, the process was reduced by approximately 93%. Taking into account these observations, it is clear from our data that the muscular dysgenesis mouse provides a relevant model to study myoblast fusion and that m-calpain is involved in this process.     

Activation of myosin synthesis in fusing and mononucleated myoblasts

The synthesis of the heavy chain subunit of myosin has been studied in breast muscle myoblasts from embryos of the Japanese quail, Coturnix coturnix japonica, during differentiation of these cells in culture. Specifically, these experiments were done to examine the roles of myoblast fusion and the regulation of myoblast cell division in the control of myosin heavy chain synthesis.The rates of myosin heavy chain synthesis have been quantitated in cultures of fusing myoblasts by measurement of the incorporation of radioactive leucine and valine precursors into myosin heavy chain, and simultaneous determination of the intracellular specific activities of these radioactive amino acids. These measurements demonstrate that, prior to fusion, dividing myoblasts synthesize little, if any, myosin heavy chain, but that during the period of myoblast fusion, myosin heavy chain synthesis becomes activated at least 50 to 100-fold. Myosin heavy chain synthesis was also measured in mononucleated myoblasts inhibited from fusing by the presence of EGTA in the culture medium. These experiments demonstrate that myosin synthesis can be activated in mononucleated myoblasts to reach rates similar to those attained in fused myoblasts. This activation occurs under conditions in which EGTA-inhibited myoblasts were induced to withdraw from the cell division cycle by reducing the concentrations of the serum and embryo extract components of the culture medium or by prior “conditioning” of standard growth medium.These experiments, therefore, establish that the activation of myosin synthesis in breast muscle myoblasts does not require fusion, but indicate that activation is co-ordinated with the withdrawal of myoblasts from the cell division cycle.     

Role of Ca and Ca-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L₆. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca²⁺ transport into myoblasts. We have also demonstrated that a Ca²⁺-activated protease, CAP (mM), appears to relocate in response to the Ca²⁺ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca²⁺ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Caspase-1 activity is required for neuronal differentiation of PC12 cells: cross-talk between the caspase and calpain systems

Previously, we have found that caspase-1 activity is increased during myoblast differentiation to myotubes. Here we show that caspase-1 activity is required for PC12 differentiation to neuronal-like cells. Caspase-1 is shown to be activated (by immunoblotting and by assessing activity in cell extracts) in the PC12 cells following the initial stage of differentiation. The inhibition of caspase-1 arrests PC12 cells at an intermediate stage of differentiation and prevents neurite outgrowth in these cells; the inhibition is reversed upon the removal of the inhibitor. Calpastatin (calpain endogenous specific inhibitor, and a known caspase substrate) is diminished at the later stages of PC12 cell differentiation, and diminution is prevented by caspase-1 inhibition. The degradation of fodrin (a known caspase and calpain substrate) is found in the advanced stage of differentiation. Caspase-1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase-1 in two distinct differentiation processes (myoblast fusion and neuronal differentiation of PC12 cells) indicates a function for this caspase in differentiation processes, and suggests some common mechanisms underlying caspase roles in such processes.     

Cardiac high molecular weight calmodulin-binding protein is homologous to calpastatin I and calpastatin II

Calpastatin is an endogenous inhibitor of calpain, which has been implicated in various physiological and pathological processes. In the present study we determined the molecular and inhibitory properties of HMWCaMBP, calpastatin I, and calpastatin II. Western blot analysis with antibodies raised against either full length HMWCaMBP or internal peptides that are common to all isoforms showed that all three homologs have common antigenic epitopes. However, additional Western blot analysis with N-terminal specific antibodies showed that all three proteins are different at the N-terminal end. HMWCaMBP is clearly different from two other homologues, calpastatin I and II, at the N-terminal end. In addition, HMWCaMBP also showed the same affinities for m-calpain as calpastatin I and calpastatin II. Our findings suggest that HMWCaMBP is the homolog of calpastatin and may be a CaM-binding form of calpastatin.     

EXTRA-LYSOSOMAL PROTEOLYSIS AND EXPRESSION OF CALPAINS AND CALPASTATIN IN CULTURED THYROID CELLS

Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl-Leu-Tyr-7-amino-4-methylcoumarin. The Ca²⁺-independent proteolytic activity was largely inhibited by substances known to affect cysteine- and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine- and aspartic proteases. Addition of Ca²⁺did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chomatography and Western blotting demonstrated the presence of both m-calpain (40% of total calpain) and μ-calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m-calpain had a diffuse cytoplasmic distribution whereas μ-calpain was located both in the cytoplasm and at the cell—cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.     

Calpain and myogenesis: development of a convenient cell culture model

Previous studies have led us to hypothesize that m-calpain plays a pivotal role in myoblast fusion through its involvement in cell membrane and cytoskeleton component reorganization. To support this hypothesis, a convenient and simple myoblast culture model using frozen embryonic myoblasts was developed, which resolved a number of problems inherent to cell primary culture. Biological assays on cultured myoblasts using different media to define the characteristics of the fusion process were first conducted. Proteinase was detectable before the initiation of the fusion process and was closely correlated to the phenomenon of fusion under each culture condition studied. In addition, the study of calpastatin showed that the initiation of fusion does not require a decrease in the level of this endogenous inhibitor of calpains and also confirmed that calpastatin may be implicated in the determination of the end of fusion. On the other hand, analysis of the evolution of myogenic factors revealed that myogenins, MyoD and Myf5, increase very significantly during the formation of multinucleated myotubes. Moreover, the antisense technique against myogenin is capable of preventing the process of fusion by 50%, confirming the pivotal role of this factor in the early stages of differentiation. The possible role of myogenic regulator factors on m-calpain gene expression is discussed.     

Identification of μ-, m-calpains and calpastatin and capture of μ-calpain activation in endothelial cells

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 μM Ca²⁺, comparable to the Ca²⁺ requirement of m-calpain. By Western blot analysis, the large subunit of μ-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of μ- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of μ-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of μ-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of μ-calpain, while the fully autolyzed postautolysis form of μ-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that μ-calpain may be involved in endothelial cell function mediated by Ca²⁺ via the limited proteolysis of various proteins. J. Cell. Biochem. 66:197-209, 1997. © 1997 Wiley-Liss, Inc.     

Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27

Evidence is presented that calpain, a calcium-activated protease, degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation. Calpain activity is required during an early stage of the adipocyte differentiation program. Thus, inhibition of calpain with N-acetyl-Leu-Leu-norleucinal (ALLN) blocks clonal expansion and acquisition of the adipocyte phenotype only when added between 12 and 24 h after the induction of differentiation. Likewise, inhibition of calpain by overexpression of calpastatin, the specific endogenous inhibitor of calpain, prevents 2-day post-confluent preadipocytes from reentering the cell cycle triggered by the differentiation inducers. Inhibition of calpain with ALLN causes preadipocytes to arrest just prior to S phase and prevents phosphorylation of the retinoblastoma gene product, DNA replication, clonal expansion, and subsequent adipocyte differentiation but does not affect the expression of immediate early genes (i.e. fos, jun, C/EBPbeta, and C/EBPdelta). Inhibition of calpain by either ALLN or by overexpression of calpastatin blocks the degradation of p27. p27 is degraded in vitro by cell-free extracts from clonally expanding preadipocytes that contain "active" calpain but not by extracts from pre-mitotic preadipocytes that do not. This action is inhibited by calpastatin or ALLN. Likewise, p27 in preadipocyte extracts is a substrate for purified calpain; this proteolytic action was inhibited by heat inactivation, EGTA, or ALLN. Thus, extracellular signals from the differentiation inducers appear to activate calpain, which degrades p27 allowing density-dependent inhibited preadipocytes to reenter the cell cycle and undergo mitotic clonal expansion.     

Constant expression of mouse calpastatin isoforms during differentiation in myoblast cell line, C2C12

C2C12 is a myoblast cell line which is used to studydifferentiation into multinucleated cells in vitro. Addition of calpain inhibitors, calpeptin orE-64d, to the culture medium prevented the myoblasticfusion of C2C12 cells. Immunoblot studies usingaffinity-purified antibody, revealed that the expressedlevels of mouse calpastatin remained unaltered duringC2C12 cell fusion. The detected calpastatin migratedas a protein of 130 kDa on SDS-polyacrylamide gelelectrophoresis. The estimated molecular mass wassomewhat greater than that in mouse liver anderythrocytes, and much greater than that reported inrat myoblasts. The 130 kDa isoform may contain anadditional N-terminal region designated XL domainfound in bovine calpastatin.