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d-β-Hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a membrane enzyme, has been purified to homogeneity from dromedary (Camelus dromedarius) liver mitochondria. Our new purification method consisted of the solubilization of mitochondrial membranes by Triton X 100 and purification of BDH by two steps: DEAE-Sephacel and Phenyl-Sepharose. The molecular mass of the enzyme subunit size was 67 kDa. The purified enzyme is recognized by anti rat liver mitochondrial BDH antibodies. Furthermore, BDH activity was absolutely dependent upon phospholipids. BDH is also characterized by specific enzymatic parameters: an optimum pH of approximately 8 for the oxidation reaction, and approximately 7 for the reduction reaction and kinetic constant (Michaelis and dissociation constants) values of 1.07±0.13 mM for KMBOH, 0.21±0.01 mM for KMNAD+, 1.04±0.20 mM for KDNAD+, 0.29±0.01 mM for KMAcAc, 0.27±0.03 mM KMNADH and 1.12±0.18 mM for KDNADH. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 24months 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. [Copyright &y& Elsevier]

Abstract: Recent research carried out in our laboratory has shown that IGF-1, TGF-β1, and insulin were able to strongly stimulate myoblast migration by increasing milli-calpain expression and activity. However, the signalling pathways involved in these phenomena remain unknown. The aim of this study was to identify the signalling pathway(s) responsible for the effects of IGF-1, TGF-β1, and insulin on myoblast migration and on milli-calpain expression and activity. For this purpose, wound healing assays were carried out in the presence of growth factors with or without specific inhibitors of ERK/MAP kinase and PI3K/Akt pathways. The results clearly showed that the inhibition of the ERK/MAP kinase pathway prevents the effects of growth factors on myoblast migration. Secondly, the expression and the activity of milli-calpain were studied in cells treated with growth factor, alone or with ERK/MAP kinase inhibitor. The results demonstrated that the up-regulation of milli-calpain expression and activity was mediated by the ERK/MAP kinase pathway. Finally, the possible implication of MyoD and myogenin, myogenic regulatory factors able to regulate milli-calpain expression, was studied. Taken together our results clearly showed that the ERK/MAP kinase signalling pathway is responsible for the effects of the three growth factors on myoblast migration and on milli-calpain expression and activity. On the opposite, the PI3K/Akt signalling pathway, MyoD and myogenin seem to be not implicated in these phenomena. [Copyright &y& Elsevier]

Abstract: Previous research in our laboratory has already shown the importance of the role played by ubiquitous calpains during myoblast migration. The aim of this study was to investigate calpain expression during myoblast migration and, to enhance this phenomenon via calpain stimulation. Ubiquitous calpains are members of a large family of calcium-dependent cysteine proteases. They play an important role in numerous biological and pathological phenomena, such as signal transduction, apoptosis, cell-cycle regulation, cell spreading, adhesion, invasion, myogenesis, and motility. Myoblast migration is a crucial step in myogenesis, as it is necessary for myoblast alignment and fusion to form myotubes. This study started by examining changes in calpain expression during migration, then investigated the possibility of activating myoblast migration via the stimulation of calpain expression and/or activity. The migration rate of myoblasts overexpressing mu- or milli-calpain was quantified. The results showed that calpain overexpression dramatically inhibited myoblast migration. Growth-factor treatments were then used to enhance myoblast migration. The results showed that treatment with IGF-1, TGF-β1, or insulin induced a major increase in migration and caused a significant increase in m-calpain expression and activity. The increase in migration was totally inhibited by adding calpeptin, a calpain-specific inhibitor. These findings suggest that milli-calpain is involved in growth factor-mediated migration. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]