Calpain concentration is elevated although net calcium-dependent proteolysis is suppressed in dystrophin-deficient muscle.

The concentration, activity, and distribution of calcium-dependent proteases (calpains) are compared in dystrophin-deficient (mdx) and control mouse muscle. Calpains have been implicated previously as the protease responsible for the observed necrosis in dystrophin-deficient human muscle. Although these mouse and human muscular dystrophies have been attributed to similar genetic defects, the mouse dystrophy shows a brief necrotic episode while the human deficiency results in progressive, lethal muscle necrosis. Findings of the present study show that control mouse muscle contains more calcium-dependent proteolytic activity than dystrophin-deficient muscle. Paradoxically, adult, dystrophin-deficient mouse muscle contains higher concentrations of calpain than found in controls. Furthermore, indirect immunofluorescence using antisera produced against an oligopeptide found in the proteolytic domain of calpain shows that calpain distribution in dystrophin-deficient muscle is dispersed throughout the cytoplasm while immunolabeling of control muscle shows calpain concentrated at Z-discs. This redistribution is consistent with calpain activation in dystrophic muscle. These findings indicate that mdx mice possess the capability of suppressing calpain-mediated proteolysis. We speculate that this suppression may enable dystrophin-deficient mouse muscle to arrest necrosis and regenerate successfully.     

Involvement of inositol 1,4,5-trisphosphate in nicotinic calcium responses in dystrophic myotubes assessed by near-plasma membrane calcium measurement

In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the cells followed by a fast refilling/release phase from the sarcoplasmic reticulum. In addition and unexpectedly, the inositol 1,4,5-trisphosphate receptor pathway was involved in these calcium signals only in the dystrophic myotubes. This surprising involvement of this calcium release channel in the excitation-contraction coupling could open new ways for understanding exercise-induced calcium increases and downstream muscle degeneration in mdx mice and, therefore, in DMD.     

Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis-associated IkappaBalpha/nuclear factor kappaB pathway perturbation in mice

Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.     

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca²⁺ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Calpain 3: a key regulator of the sarcomere?

Calpain 3 is a 94-kDa calcium-dependent cysteine protease mainly expressed in skeletal muscle. In this tissue, it localizes at several regions of the sarcomere through binding to the giant protein, titin. Loss-of-function mutations in the calpain 3 gene have been associated with limb-girdle muscular dystrophy type 2A (LGMD2A), a common form of muscular dystrophy found world wide. Recently, significant progress has been made in understanding the mode of regulation and the possible function of calpain 3 in muscle. It is now well accepted that it has an unusual zymogenic activation and that cytoskeletal proteins are one class of its substrates. Through the absence of cleavage of these substrates, calpain 3 deficiency leads to abnormal sarcomeres, impairment of muscle contractile capacity, and death of the muscle fibers. These data indicate a role for calpain 3 as a chef d'orchestre in sarcomere remodeling and suggest a new category of LGMD2 pathological mechanisms.     

Muscular Dystrophy in mdx Mice Despite Lack of Neuronal Nitric Oxide Synthase

Abstract: Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin-deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin-deficient mice. Thus, histological analyses of nNOS-dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.     

Calpains and muscular dystrophies

Calpains are a ubiquitous, well-conserved family of calcium-dependent, cysteine proteases. Their function in muscle has received increased interest because of the discoveries that the activation and concentration of the ubiquitous calpains increase in the mouse model of Duchenne muscular dystrophy (DMD), but null mutations of muscle specific calpain causes limb girdle muscular dystrophy 2A (LGMD2A). These findings indicate that modulation of calpain activity contributes to muscular dystrophies by disrupting normal regulatory mechanisms influenced by calpains, rather than through a general, nonspecific increase in proteolysis. Thus, modulation of calpain activity or expression through pharmacological or molecular genetic approaches may provide therapies for some muscular dystrophies.     

MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy

miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.     

Alteration in calcium handling at the subcellular level in mdx myotubes

In this study, we have tested the hypothesis that augmented [Ca(2+)] in subcellular regions or organelles, which are known to play a key role in cell survival, is the missing link between Ca(2+) homeostasis alterations and muscular degeneration associated with muscular dystrophy. To this end, different targeted chimeras of the Ca(2+)-sensitive photoprotein aequorin have been transiently expressed in subcellular compartments of skeletal myotubes of mdx mice, the animal model of Duchenne muscular dystrophy. Direct measurements of the [Ca(2+)] in the sarcoplasmic reticulum, [Ca(2+)](sr), show a higher steady state level at rest and a larger drop after KCl-induced depolarization in mdx compared with control myotubes. The peaks in [Ca(2+)] occurring in the mitochondrial matrix of mdx myotubes are significantly larger than in controls upon KCl-induced depolarization or caffeine application. The augmented response of mitochondria precedes the alterations in the Ca(2+) responses of the cytosol and of the cytoplasmic region beneath the membrane, which become significant only at a later stage of myotube differentiation. Taking into account the key role played by mitochondria Ca(2+) handling in the control of cell death, our data suggest that mitochondria are potential targets of impaired Ca(2+) homeostasis in muscular dystrophy.     

Degradation of skeletal muscle plasma membrane proteins by calpain

Summary Observations described here provide the first demonstration that calpain (Ca²⁺-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca²⁺) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca²⁺) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83-and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca²⁺, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0°C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane.     

Calpains in muscle wasting

Calpains are intracellular nonlysosomal Ca(2+)-regulated cysteine proteases. They mediate regulatory cleavages of specific substrates in a large number of processes during the differentiation, life and death of the cell. The purpose of this review is to synthesize our current understanding of the participation of calpains in muscle atrophy. Muscle tissue expresses mainly three different calpains: the ubiquitous calpains and calpain 3. The participation of the ubiquitous calpains in the initial degradation of myofibrillar proteins occurring in muscle atrophy as well as in the necrosis process accompanying muscular dystrophies has been well characterized. Inactivating mutations in the calpain 3 gene are responsible for limb-girdle muscular dystrophy type 2A and calpain 3 has been found to be downregulated in different atrophic situations, suggesting that it has to be absent for the atrophy to occur. The fact that similar regulations of calpain activities occur during exercise as well as in atrophy led us to propose that the calpains control cytoskeletal modifications needed for muscle plasticity.     

Localization of calpain 3 in human skeletal muscle and its alteration in limb-girdle muscular dystrophy 2A muscle

Calpain 3/p94, the skeletal muscle-specific isoform of the calpain large subunit family, is a protein product of the gene responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Through yeast two-hybrid experiments, calpain 3 has been shown to bind to titin in myofibrils [Sorimachi et al. (1995) J. Biol. Chem. 270, 31158-31162]. However, because of extensive autolysis activity, calpain 3 localization in skeletal muscle has been undefined. In this study, we generated a polyclonal antibody against an N-terminal 98-amino-acid calpain 3 fragment, which is not homologous to the corresponding regions of other conventional calpains. This antibody stained myofibrils with a unique repeated doublet-pattern. Confocal microscopic observation with marker antibodies confirmed that calpain 3 is localized in the N2 region of myofibrils. Furthermore, using this antibody, we examined the localization of calpain 3 in LGMD2A muscles.     

Calpains (intracellular calcium-activated cysteine proteinases): structure-activity relationships and involvement in normal and abnormal cellular metabolism

1. Calpains (calcium-activated cysteine proteinases) have evolved by gene fusion events involving calmodulin-like genes, cysteine proteinase genes and other sequences of unknown origin. 2. The enzymes are composed of two non-identical subunits, each of which contains functional calcium-binding sequences. 3. Calpains are inhibited by the endogenous protein inhibitor, calpastatin and some calmodulin antagonists are also inhibitors of calpain. A number of synthetic proteinase inhibitors also inhibit calpains. 4. Calpains can be activated by phospholipids, an endogenous protein activator and some amino acid derivatives. 5. Various protein substrates for calpains have been recognized in vitro, but the identity of in situ substrates remains unclear. 6. Proposals have been made for calpain function, including involvement in signal transduction, platelet activation, cell fusion, mitosis and cytoskeleton and contractile protein turnover. 7. Calpain and calpastatin expression is altered in a number of abnormal states including muscular dystrophy, muscle denervation and tenotomy, hypertension and platelet abnormalities.     

Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.     

The weak combined magnetic field,tuned to ion parametric resonance for Ca<Superscript>2+</Superscript>, stimulates dystrophin synthesis in the skeletal muscles of mdx mice subjected to cell therapy

The possibility of using bone marrow stem cells for treatment of Duchenne muscular dystrophy is intensely studied. Mdx mice are the most widely used laboratory model of Duchenne muscular dystrophy. One approach of cell therapy of muscular dystrophy is substitution of bone marrow in mdx mice after their X-ray irradiation. However, this method does not allow one to increase significantly dystrophin synthesis in muscular fibers of mdx mice. To improve the effect of transplanted cells on muscle regeneration, we additionally treated mdx mice subjected to transplantation of bone marrow cells with a weak combined magnetic field tuned to ion parametric resonance for Ca²⁺ (Ca²⁺-CMF). We found that, in irradiated chimeric 3 and 5 Gy mdx mice, additional treatment with Ca²⁺-CMF for 1 month resulted in significant increases in the portions of dystrophin-positive muscle fibers, by 15.8 and 18.3%, respectively, as compared to the control groups. Furthermore, the share of muscle fibers without centrally located nuclei also increased. We suggest that the magnetic field with these parameters may stimulate functioning of nuclei of donor cells, which were incorporated into muscle fibers.     

Calpain 3 is activated through autolysis within the active site and lyses sarcomeric and sarcolemmal components

Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.     

Calpain

The calcium-dependent thiol proteases, calpains, are widely expressed with ubiquitous and tissue specific isoforms. Calpains have been implicated in basic cellular processes including cell proliferation, apoptosis and differentiation. The focus of the current review is to summarize recent findings implicating calpains in cytoskeletal rearrangements and cell migration. Calpain cleaves many cytosolic proteins and therefore to be effective and limited in its scope, calpain activity has to be tightly regulated both temporally and spatially. Some mechanisms of regulation include calcium, growth factor-mediated phosphorylation and membrane targeting. Calpain inhibition reduces migration rates and inhibits cell invasiveness. Two putative mechanisms of calpain action during migration include its role as a signaling intermediate, acting upstream of Rho, and its effects on focal adhesion structure and disassembly. Therefore, calpains and downstream signaling molecules may be future targets for therapeutic interventions to treat cancer or chronic inflammation.     

Calpain activity in fast, slow, transforming, and regenerating skeletal muscles of rat

Fiber-type transitions in adult skeletal muscleinduced by chronic low-frequency stimulation (CLFS) encompasscoordinated exchanges of myofibrillar protein isoforms. CLFS-inducedelevations in cytosolic Ca²⁺ could activate proteases,especially calpains, the major Ca²⁺-regulated cytosolicproteases. Calpain activity determined by a fluorogenic substrate inthe presence of unaltered endogenous calpastatin activities increasedtwofold in low-frequency-stimulated extensor digitorum longus (EDL)muscle, reaching a level intermediate between normal fast- andslow-twitch muscles. µ- and m-calpains were delineated by acalpain-specific zymographical assay that assessed total activitiesindependent of calpastatin and distinguished between native andprocessed calpains. Contrary to normal EDL, structure-bound, namelymyofibrillar and microsomal calpains, were abundant in soleus muscle.However, the fast-to-slow conversion of EDL was accompanied by an earlytranslocation of cytosolic µ-calpain, suggesting that myofibrillarand microsomal µ-calpain was responsible for the twofold increase inactivity and thus involved in controlled proteolysis during fibertransformation. This is in contrast to muscle regeneration wherem-calpain translocation predominated. Taken together, we suggest thattranslocation is an important step in the control of calpain activityin skeletal muscle in vivo.