André Ducastaing

Ca2+-dependent proteinases (calpains) and muscle cell differentiation

Abstract 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-β (TGF-β) 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.

Ca2+-dependent association between a Ca2+-activated neutral proteinase (CaANP) and its specific inhibitor.

A specific inhibitor of the calcium-activated neutral proteinase (CaANP) detected first in [ 1 ] was subsequently purified from rabbit skeletal muscle [2] and bovine cardiac muscle [3]. When we have purified CaANP from rabbit skeletal muscle, no calcium-stimulated proteolytic activity on casein could be detected in the crude extract but after sepuation from the inhibitor by ion-exchange chromatography, this activity was easily measured. The presence of a similar proteinase inhibitor was also demonstrated in various other tissues (brain, lung, heart, liver) [4-6]. The simultaneous presence of CaANP [5-lo] in these tissues seems to indicate that proteolysis by this enzyme may be closely controlled. Ca2+ may play a role in the association between the enzyme and its specific inhibitor [3,5,11]. CaANP was made from the homogenate according to our standard procedure up to the Szoo step [ 121. Consequently, the enzyme still contained one 73 000 M, and two 30 000 Mr polypeptide chains. Casein (Merck) was used as a substrate to quantitate proteolytic activity as in [ 121.

Free calcium and calpain I activity

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.

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.

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.

Association of calpains 1 and 2 with protein kinase C activities.

Calpains 1 and 2 co‐eluted with protein kinase C activities after hydrophobic (phenyl‐Sepharose) and anion‐exchange (Mono Q) chromatographies of a 100 000 × g supernatant which was defined as cytosol. After centrifugation of the cytosol at 200 000 × g for 16 h, the major part of calpain 1 and of its associated protein kinase C activity was recovered in the pellet, when the major part of calpain 2, also associated to a protein kinase C activity, was present in the resulting supernatant. Polyacrylamide gel electrophoresis of the fractions eluted from the Mono Q column, which contained calpains 1 or 2 and their associated protein kinase C activities, revealed two main bands with a molecular mass of 80 and 28 kDa.

Isolation and identification of a μ-calpain-protein kinase Cα complex in skeletal muscle

A μ‐calpain‐PKC complex was isolated from rabbit skeletal muscle by ultracentrifugation and by anion‐exchange chromatography. The PKC associated to μ‐calpain was stimulated by calcium, phosphatidylserine and diacylglycerol, and corresponds to a conventional PKC (cPKC). This complex presents an apparent molecular mass close to 190 kDa and is composed of one μ‐calpain molecule and of one cPKC molecule. Using monoclonal antibodies specific for the different cPKC isoforms, the isoenzyme associated to μ‐calpain was identified as cPKCα. Immunofluorescence staining reveals a co‐localization of μ‐calpain and cPKCα on the muscle fibre plasma membranes.

Desmin degradation and Ca2+-dependent proteolysis during myoblast fusion

Summary— It has already been reported that, in vitro, intermediate filaments such as desmin and vimentin are very susceptible to proteolysis by calpains (Ca2+‐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.

Protein kinase Cα is a calpain target in cultured embryonic muscle cells

Previously we isolated a μ-calpain/PKCα 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 PKCα 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 PKCα 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 PKCα content in the particulate fraction and the appearance of PKMα fragment. These results suggest a possible calpain dependent down-regulation process of PKCαa in our model at the time of intense fusion.In our experimental conditions phorbol myristate acetate (PMA) induced a rapid depletion of pkcα 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 PKCα, 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 μ-calpains).Taken together, our findings argue for an involvement of calpains in the differentiation of embryonic myoblasts by limited proteolytic cleavage of PKCα.

Evidence for non-competitive inhibition between two calcium-dependent activated neutral proteinases and their specific inhibitor

Two muscle thiol proteinases causing partial degradation of myofibrillar constituents were isolated and purified from skeletal muscle. The two proteinases that differ significantly in calcium requirements were designated respectively high- and low-Ca2+-requiring proteinase. Both are inhibited, in vitro, by a specific inhibitor which is a protein also isolated from skeletal muscle. Experiments using carboxymethylated monomeric proteinases and inhibitor-conjugated Sepharose were carried out in order to understand the mechanism of control of the proteinases by the inhibitor. The results using increasing inhibitor concentrations show a non-competitive inhibition for both enzymes. The Ki value for the low-Ca2+-requiring form was 0.3 microM, while the Ki value for the high-Ca2+-requiring form was 0.9 microM. Likewise, the low-Ca2+-requiring form needs about 3-fold more inhibitor than the high-Ca2+-requiring form for the same per cent inhibition.