calpain and Muscular-Dystrophy--Animal

Although the exact nature of the relationship between calcium and the pathogenesis of Duchenne muscular dystrophy (DMD) is not fully understood, this is an important issue which has been addressed in several recent reviews (Alderton and Steinhardt, 2000a, Gailly, 2002, Allen et al., 2005). A key question when trying to understand the cellular basis of DMD is how the absence or low level of expression of dystrophin, a cytoskeletal protein, results in the slow but progressive necrosis of muscle fibres. Although loss of cytoskeletal and sarcolemmal integrity which results from the absence of dystrophin clearly plays a key role in the pathogenesis associated with DMD, a number of lines of evidence also establish a role for misregulation of calcium ions in the DMD pathology, particularly in the cytoplasmic space just under the sarcolemma. A number of calcium-permeable channels have been identified which can exhibit greater activity in dystrophic muscle cells, and exIsting evidence suggests that these may represent different variants of the same channel type (perhaps the transient receptor potential channel, TRPC). In addition, a prominent role for calcium-activated proteases in the DMD pathology has been established, as well as modulation of other intracellular regulatory proteins and signaling pathways. Whether dystrophin and its associated proteins have a direct role in the regulation of calcium ions, calcium channels or intracellular calcium stores, or indirectly alters calcium regulation through enhancement of membrane tearing, remains unclear. Here we focus on areas of consensus or divergence amongst the existing literature, and propose areas where future research would be especially valuable.

Topics: Animals; Calcium; Calcium Channels; Calpain; Dystrophin; Humans; Muscle, Skeletal; Muscular Dystrophy, Animal; Muscular Dystrophy, Duchenne; Sarcolemma

Topics: Animals; Calcium; Calpain; Chaperonins; Connectin; Humans; Isoenzymes; Membrane Proteins; Models, Structural; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Muscular Dystrophy, Animal; Protein Conformation; Protein Kinases; Recombinant Proteins

Duchenne muscular dystrophy (DMD) is a devastating genetic disorder that leads to compromised cellular membranes, caused by the absence of membrane-bound dystrophin protein. Muscle membrane leakage results in disrupted intracellular homeostasis, protein degradation, and muscle wasting. Improving muscle membrane integrity may delay disease progression and extend the lifespan of DMD patients. Here, we demonstrate that exosomes, membranous extracellular vesicles, can elicit functional improvements in dystrophic mice by improving muscle membrane integrity. Systemic administration of exosomes from different sources induced phenotypic rescue and mitigated pathological progression in dystrophic mice without detectable toxicity. Improved membrane integrity conferred by exosomes inhibited intracellular calcium influx and calcium-dependent activation of calpain proteases, preventing the degradation of the destabilized dystrophin-associated protein complex. We show that exosomes, particularly myotube-derived exosomes, induced functional improvements and alleviated muscle deterioration by stabilizing damaged muscle membrane in dystrophic mice. Our findings suggest that exosomes may have therapeutic implications for DMD and other diseases with compromised membranes.

Topics: Animals; Calcium; Calpain; Cell Membrane; Disease Models, Animal; Dystrophin; Exosomes; Humans; Mice; Mice, Inbred mdx; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Animal; Muscular Dystrophy, Duchenne; Peptide Hydrolases

Previous studies have tested the hypothesis that calpain and/or proteasome inhibition is beneficial in Duchenne muscular dystrophy, based largely on evidence that calpain and proteasome activities are enhanced in the mdx mouse.. mRNA expression of ubiquitin-proteasome and calpain system components were determined using real-time polymerase chain reaction in skeletal muscle and heart in the golden retriever muscular dystrophy model. Similarly, calpain 1 and 2 and proteasome activities were determined using fluorometric activity assays.. We found that less than half of the muscles tested had increases in proteasome activity, and only half had increased calpain activity. In addition, transcriptional regulation of the ubiquitin-proteasome system was most pronounced in the heart, where numerous components were significantly decreased.. This study illustrates the diversity of expression and activities of the ubiquitin-proteasome and calpain systems, which may lead to unexpected consequences in response to pharmacological inhibition.

Topics: Animals; Calpain; Disease Models, Animal; Dogs; Gene Expression Regulation; Muscle, Skeletal; Muscular Dystrophy, Animal; Myocardium; Proteasome Endopeptidase Complex; Ubiquitin; Ubiquitin-Protein Ligase Complexes

Proteolytically active calpain-3/p94 is clearly vital for normal muscle function, since its absence leads to limb girdle muscular dystrophy 2A, but its function and regulatory control are poorly understood. Here we use single muscle fibers, individually skinned by microdissection, to investigate the diffusibility and autolytic activation of calpain-3 in situ. Virtually all calpain-3 present in mature muscle fibers is tightly bound in the vicinity of the titin N2A line and triad junctions and remains so irrespective of fiber stretching or raised [Ca(2+)]. Most calpain-3 is evidently bound within the contractile filament lattice, because (i) its slow diffusional loss is slowed further by locking myosin and actin into rigor and (ii) detergent dispersion of membranes causes rapid washout of most ryanodine receptors and sarcoplasmic reticulum Ca(2+) pumps with little accompanying washout of calpain-3. Calpain-3 autolyzes (becoming proteolytically active) in a tightly calcium-dependent manner. It remains in its nonactivated full-length form if [Ca(2+)] is maintained at < or = 50 nm, the normal resting level, even with brief increases to 2-20 mum during repeated tetanic contractions, but it becomes active (though still bound) if [Ca(2+)] is kept slightly elevated at 200 nm ( approximately 20% autolysis in 1 h). Calpain-3 did not spontaneously autolyze even when free in solution with 200 nm Ca(2+) for up to 60 min. These findings explain why calpain-3 remains quiescent with normal exercise but is activated following eccentric (stretching) contractions, when resting [Ca(2+)] is elevated, and how a protease such as calpain-3 can be very Ca(2+)-sensitive yet highly specific in its actions.

Topics: Animals; Calcium; Calcium Channels; Calpain; Connectin; Cytoplasm; Dose-Response Relationship, Drug; Isoenzymes; Male; Muscle Contraction; Muscle Fibers, Skeletal; Muscle Proteins; Muscular Dystrophy, Animal; Organ Culture Techniques; Physical Conditioning, Animal; Rats; Rats, Long-Evans; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

BN 82270 is a membrane-permeable prodrug of a chimeric compound (BN 82204) dually acting as calpain inhibitor and anti-oxidant. Acute in vivo injection of dystrophic mdx mice (30 mg/kg, s.c.) fully counteracted calpain overactivity in diaphragm. A chronic 4-6 weeks administration significantly prevented in vivo the fore limb force drop occurring in mdx mice exercised on treadmill. Ex vivo electrophysiological recordings showed that BN 82270 treatment contrasted the decrease in chloride channel function (gCl) in diaphragm, an index of spontaneous degeneration, while it was less effective on both exercise-impaired gCl and calcium-dependent mechanical threshold of the hind limb extensor digitorum longus (EDL) muscle fibres. The BN 82270 treated mdx mice showed a marked reduction of plasma creatine kinase and of the pro-fibrotic cytokine TGF-beta1 in both hind limb muscles and diaphragm; however, the histopathological profile of gastrocnemious muscle was poorly ameliorated. In hind limb muscles of treated mice, the active form was detected by HPLC in the low therapeutic concentration range. In vitro exposure to 100 microM BN 82270 led to higher active form in diaphragm than in EDL muscle. This is the first demonstration that this class of chimeric compounds, dually targeting pathology-related events, exerts beneficial effects in muscular dystrophy. The drug/prodrug system may require posology adjustment to produce wider beneficial effects on all muscle types.

Topics: Animals; Antioxidants; Biomechanical Phenomena; Body Weight; Calpain; Chloride Channels; Creatine Kinase; Diaphragm; Glycoproteins; Hindlimb; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Animal; Phenothiazines; Physical Conditioning, Animal; Prodrugs; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1

Dipeptide-derived alpha-keto-amide compounds with potent calpain inhibitory activity have been identified. These reversible covalent inhibitors have IC(50) values down to 25nM and exhibit greatly improved activity in muscle cells compared to the reference compound MDL28170. Several novel calpain inhibitors have shown positive effects on histological parameters in an animal model of Duchenne muscular dystrophy demonstrating their potential as a treatment option for this fatal disease.

Topics: Amides; Animals; Calpain; Cysteine Proteinase Inhibitors; Diaphragm; Dipeptides; Mice; Mice, Inbred mdx; Molecular Structure; Muscular Dystrophy, Animal

The giant protein titin serves a primary role as a scaffold for sarcomere assembly; however, proteins that mediate this remodeling have not been identified. One potential mediator of this process is the protease calpain 3 (C3), the protein mutated in limb girdle muscular dystrophy type 2A. To test the hypothesis that C3 mediates remodeling during myofibrillogenesis, C3 knockout (C3KO) mice were generated. The C3KO mice were atrophic containing small foci of muscular necrosis. Myogenic cells fused normally in vitro, but lacked well-organized sarcomeres, as visualized by electron microscopy (EM). Titin distribution was normal in longitudinal sections from the C3KO mice; however, EM of muscle fibers showed misaligned A-bands. In vitro studies revealed that C3 can bind and cleave titin and that some mutations that are pathogenic in human muscular dystrophy result in reduced affinity of C3 for titin. These studies suggest a role for C3 in myofibrillogenesis and sarcomere remodeling.

Topics: Animals; Calpain; Connectin; Mice; Mice, Knockout; Muscle Development; Muscle Proteins; Muscular Dystrophies, Limb-Girdle; Muscular Dystrophy, Animal; Protein Kinases; Sarcomeres

Reduced sarcolemmal integrity in dystrophin-deficient muscles of mdx mice and Duchenne muscular dystrophy (DMD) patients has been reported to result in altered calcium homeostasis. Previous studies have shown a correlative relationship between calcium-dependent protease (calpain) activity in dystrophic muscle and muscle necrosis, but have not tested whether calpain activation precedes cell death or is a consequence of it. To test a causal relationship between calpain activation and muscle cell death in dystrophin deficiency, mdx mice were generated that overexpress a calpastatin transgene in muscle. Calpastatin (CS) is a specific, endogenous inhibitor of m- and micro -calpains that does not inhibit calpain 3 (p94). CS overexpression on a C57/BL 10 background produced no phenotype. Transgenic (Tg) mice crossed with mdx mice were tested for pathological indicators of necrosis, regeneration and membrane damage. Two lines of mice were examined, with different levels of CS overexpression. Both lines of Tg/mdx mice showed reductions in muscle necrosis at 4 weeks of age. These mice had fewer as well as smaller lesions. In addition, one line of mice had significantly less regeneration, indicating a reduction in previous necrosis. The extent of improvement correlated with the level of CS protein expression. Membrane damage, as assessed by procion orange and creatine kinase assays, was unchanged, supporting the idea that calpains act downstream of the primary muscle defect. These data suggest that calpains play an active role in necrotic processes in dystrophic muscle and that inhibition of calpains might provide a good therapeutic option for treatment of DMD.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Membrane; Down-Regulation; Mice; Mice, Inbred mdx; Mice, Transgenic; Muscle Cells; Muscle, Skeletal; Muscular Dystrophy, Animal

Muscular dystrophy with myositis (mdm) is a recessive mouse mutation that causes severe and progressive muscular degeneration. Here we report the identification of the mdm mutation as a complex rearrangement that includes a deletion and a LINE insertion in the titin (Ttn) gene. Mutant allele-specific splicing results in the deletion of 83 amino acids from the N2A region of TTN, a domain thought to bind calpain-3 (CAPN3) the product of the human limb-girdle muscular dystrophy type 2A (LGMD2A) gene. The Ttn(mdm) mutant mouse may serve as a model for human tibial muscular dystrophy, which maps to the TTN locus at 2q31 and shows a secondary reduction of CAPN3 similar to that observed in mdm skeletal muscle. This is the first demonstration that a mutation in Ttn is associated with muscular dystrophy and provides a novel animal model to test for functional interactions between TTN and CAPN3.

Topics: Animals; Calpain; Chromosome Mapping; Cloning, Molecular; Connectin; Gene Deletion; Ligands; Mice; Mice, Inbred C57BL; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophy, Animal; Mutagenesis, Insertional; Myositis; Peptide Fragments; Protein Binding; Protein Kinases; Protein Structure, Tertiary

Labrador retrievers suffer from an autosomal recessive muscular dystrophy of unknown aetiology. Dogs affected with this disease develop generalized weakness associated with severe, generalized skeletal muscle atrophy and mild elevations in creatine kinase in the first few months of life. The severity of signs tends to progress over the first year of life but can vary from mild exercise intolerance to non-ambulatory tetraparesis. Beyond 1 year of age, the signs usually stabilize and although muscle mass does not increase, affected dogs' strength may improve slightly. The pathological changes present on muscle biopsy include marked variation in muscle fibre size with hypertrophied and round atrophied fibres present. There is an increased number of fibres with central nuclei and split fibres can be seen. It has been suggested that the disorder is a model for limb-girdle muscular dystrophy. In recent years, mutations in genes encoding the proteolytic enzyme, calpain 3, a novel protein named dysferlin, and components of the dystrophin-glycoprotein complex have been identified as causes of autosomal recessive limb-girdle muscular dystrophy. We have evaluated these proteins in normal dogs and in three Labrador retrievers with autosomal recessive muscular dystrophy using immunohistochemistry and Western blot analysis on frozen skeletal muscle. The results demonstrate that dystrophin, the sarcoglycans, alpha-actinin, dysferlin and calpain 3 are present in the normal and affected dogs. We conclude that this autosomal recessive muscular dystrophy is not due to a deficiency of alpha-actinin, or any of the known autosomal recessive limb-girdle muscular dystrophy proteins, although we cannot rule out a malfunction of any of these proteins.

Topics: Actinin; Animals; Calpain; Dog Diseases; Dogs; Dysferlin; Dystrophin; Female; Genes, Recessive; Glycoproteins; Male; Membrane Proteins; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophy, Animal

In muscular dystrophy (MD) the imbalance between muscle protein synthesis and degradation may be an important factor leading to muscle wasting. The three major pathways of muscle proteolysis identified in skeletal muscle are: the lysosomal cathepsin pathway, the calcium-dependent calpain pathway, and the ATP-dependent ubiquitin pathway. Insulin-like growth factor I (IGF-I) and a high-protein diet (HPD) have been shown to reduce proteolysis in skeletal muscle. We examined the effect of 6 weeks of recombinant human IGF-I (rhIGF-I) alone or in combination with HPD treatment on the proteolytic pathways in skeletal muscle of 129 ReJ dystrophic (dy) mice. (A group of normal (Norm) nondystrophic (129 J) mice were included as controls). Untreated dy mice exhibited increased net proteolysis (P < 0.05), elevated net calpain activity (P < 0.01), and increased ubiquitin levels when compared to control mice (P < 0.05). Our evidence suggests that HPD and rhIGF-I decrease proteolysis in the 129 ReJ dy mouse. This effect appears attributable, at least in part, to reduced calpain-mediated myofibrillar breakdown (P < 0.05) due to decreased calpain autolysis or increased calpastatin levels. In contrast to calpain, cathepsin B activity was increased in HPD and rhIGF-I + HPD-treated dy muscle (P < 0.05) and unaltered in the rhIGF-I treated animals. Levels of free and protein-conjugated ubiquitin were also increased in rhIGF-I, and rhIGF-I + HPD treated dyanimals (P < 0.05). The amelioration of muscle wasting in the 129 ReJ dy model by HPD and/or rhIGF-I may have potential implications in the treatment of human MD.

Topics: Animals; Blotting, Western; Calpain; Cathepsin B; Dietary Proteins; Humans; Insulin-Like Growth Factor I; Male; Mice; Mice, Mutant Strains; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophy, Animal; Recombinant Proteins; Ubiquitins

Previous studies have shown that calpains are autolytically cleaved during the disease process of mdx dystrophy, a mouse model for Duchenne muscular dystrophy, indicating that calpains may be activated and play a role in proteolysis that occurs in muscular dystrophy (J. Biol. Chem. 270(18), 10909-10914, 1995). In the present study, we investigated the location of calpain in dystrophic muscle fibers over the course of mdx dystrophy, to relate the protease distribution to its state of activation, and to determine whether calpain translocation was an early event in mdx dystrophy. Immunolabeling of health, fully differentiated muscle fibers showed calpain present throughout the cytosol, but more concentrated near the plasma membrane. However, degenerating mdx fibers did not contain higher concentrations of calpain at the plasma membrane and showed only a homogeneous, cytosolic distribution. Calpain distribution was similarly diffuse in young myotubes and regenerating fibers with increased cytosolic concentration in early myotubes. Calpain distribution in adult mdx tissue was similar to that occurring in healthy, fully differentiated fibers, although adult mdx fibers displayed higher concentrations of membrane-associated calpain than those observed in C57 controls. The association of calpain with the plasma membrane was verified by immunoblots of isolated sarcolemmal membrane from adult mdx and control muscle which showed calpain present predominantly in the cytosol along with some membrane association. Thus, changes in calpain distribution coincide with changes in enzymatic cleavage over the course of mdx dystrophy shown previously. Furthermore, the stages of pathology at which calpain cleavage is least coincides with those stages when calpain is most concentrated at the cell membrane, suggesting that calpain is retained in an inactive form at the plasma membrane.

Topics: Animals; Antibody Specificity; Calpain; Cell Membrane; Cytosol; Enzyme Activation; Mice; Mice, Inbred mdx; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Animal; Necrosis; Regeneration; Sarcolemma

Death of dystrophin-deficient muscle purportedly results from increases in [Ca]in that cause the activation of calpains. We have tested whether calpains play a role in this process by assaying for changes in calpain concentration and activation in peak necrotic mdx mice (4 weeks of age) and in completely regenerated mdx mice (14 weeks of age). Biochemical fractionation and immunoblotting with epitope-specific antisera allowed measurement of the concentrations of m- and mu-calpains and the extent of autoproteolytic modification. Our findings show that total calpain concentration is elevated in both 4-week and 14-week mdx mice. This increase in concentration was shown to result primarily from a significant increase in m-calpain concentration at 4 weeks. Northern analysis demonstrated that neither m- nor mu-calpain mRNA concentrations differed between mdx and controls suggesting that the increased calpain concentration results from post-translational regulation. Immunoblotting with antibodies directed against amino-terminal peptides revealed an increase in autoproteolysis of mu-calpain, indicative of increased activation. The extent of autoproteolysis of mu-calpain returns to control levels during regeneration. This is not a consequence of increased calpastatin mRNA or protein. The findings reported here support a role for calpains in both the degenerative and regenerative aspects of mdx dystrophy.

Topics: Age Factors; Animals; Calcium-Binding Proteins; Calpain; Dystrophin; Enzyme Activation; Gene Expression; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscular Dystrophy, Animal; Necrosis; RNA, Messenger

Resting free calcium levels ([Ca2+]i) are elevated in Duchenne human myotubes and mdx mouse muscle and myotubes which lack the gene product dystrophin at the sarcolemma. Increased net muscle protein degradation has been directly related to this elevated [Ca2+]i. The [Ca2+]i rise may result from increased calcium influx via leak channels, which have increased opening probabilities (Po) in dystrophic cells. Dystrophin, therefore, might directly regulate leak channel activity. In intact mdx soleus muscles, protein degradation was reduced to normal levels by leupeptin, a thiol protease inhibitor. In muscle homogenates, leupeptin also abolished calcium-induced increases in protein degradation. When mouse myotubes were cultured in the continuous presence of leupeptin (50 microM), the elevation in mdx resting [Ca2+]i was prevented. Leak channel Po increased with age in mdx myotubes, whereas leupeptin-treated mdx leak channel opening probabilities were always lower or equal to the Po for untreated normal myotubes. These results indicate that increased leak channel activity in dystrophic muscle results in elevated [Ca2+]i levels, but also suggest that dystrophin does not directly regulate channel activity. Instead the results suggest that proteolysis may be responsible for the altered gating of calcium leak channels. The resultant increased channel Po in turn elevates [Ca2+]i, which further increases proteolytic activity in a positive feedback loop, leading to the eventual necrosis of the muscle fibers.

Topics: Animals; Calcium; Calcium Channels; Calpain; Cells, Cultured; Clenbuterol; Dose-Response Relationship, Drug; Dystrophin; Feedback; Glycoproteins; Kinetics; Leupeptins; Mice; Mice, Mutant Strains; Muscles; Muscular Dystrophy, Animal

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.

Topics: Aging; Animals; Calpain; Chickens; Dystrophin; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Kinetics; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscle Development; Muscles; Muscular Dystrophy, Animal; Reference Values

An endogenous inhibitor for calcium-activated neutral protease (CANP) from skeletal and cardiac muscles of muscular dystrophic hamsters (UMX 7.1) was compared with that from normal control animals at 4 and 10 weeks of age by Western blotting using antibody raised against CANP inhibitor. Fragmented CANP inhibitor was found in dystrophic skeletal muscles in all cases at both ages, while only intact inhibitor was detected in the skeletal muscle of the normal hamsters. A total absence of intact inhibitor was shown in one 10-week-old dystrophic hamster. In contrast, there was little difference in CANP inhibitor from heart between dystrophic and control hamsters at 4 weeks. However, fragmentation similar to that in skeletal muscle was seen in the heart inhibitor in a few of the 10-week-old dystrophic hamsters.

Topics: Animals; Blotting, Western; Calpain; Cricetinae; Glycoproteins; Male; Muscle Proteins; Muscles; Muscular Dystrophy, Animal; Myocardium

Topics: Animals; Calpain; Chromatography, DEAE-Cellulose; Cricetinae; Glycoproteins; Mesocricetus; Muscular Dystrophy, Animal

High molecular weight kininogen has been shown to be the principal plasma inhibitor of cellular thiol proteases including cathepsins B, H and L and calpains 1 and 2. Since these same enzymes have been reported to be elevated in animals with muscular dystrophy, we studied plasmas from hamsters with muscular dystrophy and compared these to normal hamster plasma. The ability of plasma to inhibit purified platelet calpain was assayed and found to be 62% of normal. Since low molecular weight kininogen can also inhibit calpain, the coagulant activity of kininogen, an activity unique for high molecular weight kininogen, was determined in dystrophic hamster plasma and found to be 69% of normal in close agreement with the calpain inhibitory activity. The contribution of the other plasma calpain inhibitor alpha 2-macroglobulin appeared small since inactivation with methylamine did not alter the ability to inhibit calpain in either normal or dystrophic plasma. We conclude that there is a selective deficiency of plasma high molecular weight kininogen in dystrophic hamsters, an abnormality which could play a role in the pathogenesis of this disorder.

Topics: Age Factors; alpha-Macroglobulins; Animals; Calpain; Cricetinae; Kininogens; Muscular Dystrophy, Animal; Protease Inhibitors; Whole Blood Coagulation Time

1. Two fast-twitch skeletal muscles from normal and dystrophic hamsters were analysed for their calpain and calpastatin contents. 2. Assays of wide-specificity calpain II showed that the activity levels in the two muscles were increased 1.5 and 1.6 times in dystrophic animals. 3. Analysis of calpastatin levels showed that the respective dystrophic muscles had activity levels of 2.2 and 2.8 times those of control muscles. 4. These results contrast with previous studies on denervated hamster muscles which showed that denervation causes an increase in calpain levels but a decrease in calpastatin levels.

Topics: Animals; Calcium-Binding Proteins; Calpain; Muscles; Muscular Dystrophy, Animal; Proteins

The purpose of this study was to investigate the possible role of calcium-activated neutral protease in the disorganization and dissolution of the myofibrils of the rat soleus that occurs following tenotomy. Rats were killed 3, 5, 7, 14, 21, and 42 days after tenotomy of the soleus, and the muscles were removed and assayed for calcium-activated protease activity. Maximal protease activity occurred 1 week after tenotomy, at the time when myofibril organization is completely disrupted. Activity was still high 2 and 3 weeks after the operation, but returned to normal levels by 6 weeks, when muscle histology had returned to normal. The time course of the calcium-activated protease activity corresponded closely to the time course of the morphological changes. Thus, calcium-activated neutral protease may play a major role in myofibrillar proteolysis following tenotomy and in making the myofibril susceptible to proteolytic attack by other, less specific proteases.

Topics: Animals; Calpain; Disease Models, Animal; Female; Hydrogen-Ion Concentration; Muscles; Muscular Atrophy; Muscular Dystrophy, Animal; Rats; Rats, Inbred Strains; Tendons

Calcium-activated neutral protease (milli-CANP) and its endogenous inhibitor are elevated in muscle tissues, primarily the skeletal muscle and heart, of dystrophic mice (C57BL/6J dy/dy) as compared to the control strain (C57BL/10J). Tissues showing relative increase of CANP also show significant loss of enzymes such as CK, LDH in comparison to plasma, where these enzymes register a significant increase. PK is lost minimally from these tissues, probably showing a "sparing effect." Absence of any significant change in CANP activity in the liver points to a specific role of CANP in the dystrophic process. In the skeletal muscle the endogenous CANP inhibitor registers a concomitant increase with CANP without altering the enzyme/inhibitor ratio.

Topics: Animals; Calpain; Creatine Kinase; Glycoproteins; L-Lactate Dehydrogenase; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscles; Muscular Dystrophy, Animal; Myocardium; Organ Specificity; Pyruvate Kinase; Reference Values

The relationship between proteolytic enzyme activities, soluble protein profiles, and progression of pathology in dystrophic chick muscle was investigated. Activities of cathepsins C and H, and calcium-activated protease were significantly higher in dystrophic patagialis and pectoralis muscles compared with normal muscles prior to the onset of extensive muscle fiber necrosis. Proteolytic enzyme activity of dystrophic muscle remained constant relative to normal muscle during development while muscle pathology progressed in both patagialis and pectoralis muscles. There were more protein bands (60-80 kDa) in the dystrophic muscle extracts compared with normal at all ages studied. Activities of calcium-activated protease in the dystrophic pectoralis and patagialis were similar although muscle pathology progressed much more rapidly in the dystrophic pectoralis. We conclude there is no causal relationship between the activity of the above proteolytic enzymes and the development of muscle fiber necrosis. The elevated activities of proteolytic enzymes in dystrophic muscle may be due to abnormally accelerated growth.

Topics: Animals; Calpain; Cathepsin C; Cathepsin H; Cathepsins; Chickens; Cysteine Endopeptidases; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Electrophoresis, Polyacrylamide Gel; Muscles; Muscular Dystrophy, Animal

Topics: Animals; Calpain; Chloroquine; Endopeptidases; Leupeptins; Mice; Mice, Inbred Strains; Muscles; Muscular Dystrophy, Animal; Myofibrils; Neuromuscular Junction