Patrick Cottin

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.

Myristoylated alanine-rich C kinase substrate (MARCKS) is involved in myoblast fusion through its regulation by protein kinase Cα and calpain proteolytic cleavage

MARCKS (myristoylated alanine-rich C kinase substrate) is a major cytoskeletal protein substrate of PKC (protein kinase C) whose cellular functions are still unclear. However numerous studies have implicated MARCKS in the stabilization of cytoskeletal structures during cell differentiation. The present study was performed to investigate the potential role of Ca(2+)-dependent proteinases (calpains) during myogenesis via proteolysis of MARCKS. It was first demonstrated that MARCKS is a calpain substrate in vitro. Then, the subcellular expression of MARCKS was examined during the myogenesis process. Under such conditions, there was a significant decrease in MARCKS expression associated with the appearance of a 55 kDa proteolytic fragment at the time of intense fusion. The addition of calpastatin peptide, a specific calpain inhibitor, induced a significant decrease in the appearance of this fragment. Interestingly, MARCKS proteolysis was dependent of its phosphorylation by the conventional PKCalpha. Finally, ectopic expression of MARCKS significantly decreased the myoblast fusion process, while reduced expression of the protein with antisense oligonucleotides increased the fusion. Altogether, these data demonstrate that MARCKS proteolysis is necessary for the fusion of myoblasts and that cleavage of the protein by calpains is involved in this regulation.

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

Abstract 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 μ‐ 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.

Up-regulation of calcium-dependent proteolysis in human myoblasts under acute oxidative stress

The reduced regenerative potential of muscle fibres, most likely due to a decreased number and/or function of satellite cells, could play a significant role in the progression of muscle ageing. Accumulation of reactive oxygen species has been clearly correlated to sarcopenia and could contribute to the impairment of satellite cell function. In this work we have investigated the effect of oxidative stress generated by hydrogen peroxide in cultured human skeletal muscle satellite cells. We specifically focused on the activity and regulation of calpains. These calcium-dependent proteases are known to regulate many transduction pathways including apoptosis and play a critical role in satellite cell function. In our experimental conditions, which induce an increase in calcium concentration, protein oxidation and apoptotic cell death, a significant up-regulation of calpain expression and activity were observed and ATP synthase, a major component of the respiratory chain, was identified as a calpain target. Interestingly we were able to protect the cells from these H(2)O(2)-induced effects and prevent calpain up-regulation with a natural antioxidant extracted from pine bark (Oligopin). These data strongly suggest that oxidative stress could impair satellite cell functionality via calpain-dependent pathways and that an antioxidant such as Oligopin could prevent apoptosis and calpain activation.

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.

Involvement of the calcium-dependent proteolytic system in 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 with high socio-economics implications. Although sarcopenia is well documented, the aetiology of this condition still remains poorly understood. Calpains are ubiquitous proteases regulated in part by a specific inhibitor, calpastatin. They are well known to have major implications in muscle growth and differentiation. The aim of the present study was to determine if this proteolytic system could be involved in the phenotype associated with sarcopenia. Calpains and calpastatin levels, subcellular distributions and activities were compared between muscles from 3 and 24 months old rats. Altogether, the results we obtained showed an overall increase in calpain activities associated with muscle aging. These findings suggest that the calcium-dependent proteolytic system is indeed involved in sarcopenia.

Down-regulation of MyoD by calpain 3 promotes generation of reserve cells in C2C12 myoblasts

Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated “reserve cells” are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

Involvement of micro-calpain (CAPN 1) in muscle cell differentiation.

Several studies have already demonstrated that micro- and milli-calpains (CAPN 1-CAPN 2), calcium-dependent intracellular cysteine-proteases are involved in many biological phenomenon including muscle growth and development. More particularly, recent studies have demonstrated that milli-calpain is implicated in myoblast fusion. Moreover, in primary muscle cells, these proteases do not appear simultaneously throughout muscle cell differentiation. Because micro- and milli-calpains do not have the same intracellular localization, it appears likely that these two calcium-dependent proteases have different biological roles during muscle cell differentiation. The goal of this study is to determine the role of micro-calpain. We therefore, have developed a muscle cell line in which micro-calpain is over-expressed, using the inducible Tet Regulated Expression System. The outcome is observed by following the behavior of different proteins, considered to be potential substrates of the protease. The present study shows important decreases in the expression level of ezrin (68%), vimentin (64%) and caveolin 3 (76%) whereas many other cytoskeletal proteins remain remarkably stable. Concerning the myogenic transcription factors, only the level of myogenin decreased (59%) after the over-expression of micro-calpain. Ultra structural studies have shown that the myofibrils formed near the cell periphery are normally oriented, lying along the longitudinal axis. This regularity is lost progressively towards the cell center where the cytoskeleton presented an increasing disorganization. All these results indicate that micro-calpain is involved in regulation pathway of myogenesis via at least its action on ezrin, vimentin, caveolin 3 and myogenin, a muscle transcription factor.

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.