leupeptins and Muscular-Dystrophy--Animal

It seems plausible to hypothesize that in all forms of neurodegeneration or other forms of tissue degeneration, a common pathway exists that, when deciphered, could lead to our understanding of a variety of diseases that result in tissue necrosis, as well as offer potential for therapeutic intervention. In recent years progress toward elucidating this common pathway has been accelerated through the studies of a number of laboratories, including our own, on the role of the protease calpain in this process. Thus, in a variety of disorders, such as stroke, spinal cord injury, traumatic nerve injury, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, muscular dystrophy, cataract formation, unregulated calpain proteolysis, initiated via dysregulation of calcium ion homeostasis, participates in the pathogenesis and is a potentially unifying mechanistic event. In order to demonstrate the feasibility of the approach we have taken in using the calpain inhibitor leupeptin as a therapeutic agent, I will describe two areas of research in which we have been engaged over the past 20 years. One is our long-standing interest in muscular dystrophy. The other is of more recent vintage, and involves the use of calpain inhibitors to protect sensory hair cells and spiral ganglion neurons from damage associated with acoustic trauma, this latter in collaboration with Dr. R. Salvi at SUNY-Buffalo and Dr. A. Shulman at SUNY-Downstate.

Topics: Animals; Glycoproteins; Hair Cells, Auditory; Haplorhini; Hearing Loss, Noise-Induced; Leupeptins; Mice; Mice, Inbred C57BL; Muscular Dystrophy, Animal; Nerve Degeneration; Spiral Ganglion

The microbial antiproteinases--antipain, leupeptin, and pepstatin--have been reported to inhibit the degeneration of chicken dystrophic muscle in tissue culture. Trials of antipain and pepstatin, and of leupeptin and pepstatin administered subcutaneously in murine muscular dystrophy, failed to produce evidence of benefit. It is suggested that these antiproteinases cannot pass through an intact sarcolemma into muscle fibers. Further studies with liposomes may allow these agents to enter muscle fibers.

Topics: Animals; Clinical Trials as Topic; Drug Evaluation, Preclinical; Drug Therapy, Combination; Female; Leupeptins; Male; Mice; Muscular Dystrophy, Animal; Oligopeptides; Papain; Pepstatins; Protease Inhibitors

Muscle atrophy, a significant characteristic of congenital muscular dystrophy with laminin α2 chain deficiency (also known as MDC1A), occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) plays a key role in protein degradation in skeletal muscle cells. In order to identify new targets for drug therapy against MDC1A, we have investigated whether increased proteasomal degradation is a feature of MDC1A. Using the generated dy(3K)/dy(3K) mutant mouse model of MDC1A, we studied the expression of members of the ubiquitin-proteasome pathway in laminin α2 chain-deficient muscle, and we treated dy(3K)/dy(3K) mice with the proteasome inhibitor MG-132. We show that members of the UPS are upregulated and that the global ubiquitination of proteins is raised in dystrophic limb muscles. Also, phosphorylation of Akt is diminished in diseased muscles. Importantly, proteasome inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. Specifically, treatment with MG-132 increases lifespan, enhances locomotive activity, enlarges muscle fiber diameter, reduces fibrosis, restores Akt phosphorylation and decreases apoptosis. These studies promote better understanding of the disease process in mice and could lead to a drug therapy for MDC1A patients.

Topics: Animals; Apoptosis; Blotting, Western; Cysteine Proteinase Inhibitors; Disease Models, Animal; Fluorescent Antibody Technique; Laminin; Leupeptins; Mice; Mice, Knockout; Mice, Transgenic; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophy, Animal; Phenotype; Phosphorylation; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Ubiquitin; Ubiquitination

Limb girdle muscular dystrophy type 2D (LGMD2D, OMIM600119) is a genetic progressive myopathy that is caused by mutations in the human alpha-sarcoglycan gene (SGCA). Here, we have introduced in mice the most prevalent LGMD2D mutation, R77C. It should be noted that the natural murine residue at this position is a histidine. The model is, therefore, referred as Sgca(H77C/H77C). Unexpectedly, we observed an absence of LGMD2D-like phenotype at histological or physiological level. Using a heterologous cellular model of the sarcoglycan complex formation, we showed that the R77C allele encodes a protein that fails to be delivered to its proper cellular localization in the plasma membrane, and consequently to the disappearance of a positively charged residue. Subsequently, we transferred an AAV vector coding for the human R77C protein in the muscle of Sgca-null mice and were able to pharmacologically rescue the R77C protein from endoplasmic reticulum-retention using proteasome or mannosidase I inhibitors. This suggests a therapeutic approach for LGMD2D patients carrying mutations that impair alpha-sarcoglycan trafficking.

Topics: Alkaloids; Animals; Cell Line, Tumor; Cysteine; Female; Humans; Leupeptins; Mannosidases; Mice; Mice, Knockout; Muscles; Muscular Dystrophies, Limb-Girdle; Muscular Dystrophy, Animal; Mutation, Missense; Phenotype; Protein Transport; Sarcoglycans

Duchenne muscular dystrophy (DMD) is one of the most severe X-linked, inherited diseases of childhood, characterized by progressive muscle wasting and weakness as the consequence of mutations in the dystrophin gene. The protein encoded by dystrophin is a huge cytosolic protein that links the intracellular F-actin filaments to the members of the dystrophin-glycoprotein-complex (DGC). Dystrophin deficiency results in the absence or reduction of complex components that are degraded through an unknown pathway. We show here that muscle degeneration in a Caenorhabditis elegans DMD model is efficiently reduced by downregulation of chn-1, encoding the homologue of the human E3/E4 ubiquitylation enzyme CHIP. A deletion mutant of chn-1 delays the cell death of body-wall muscle cells and improves the motility of animals carrying mutations in dystrophin and MyoD. Elimination of chn-1 function in the musculature, but not in the nervous system, is sufficient for this effect, and can be phenocopied by proteasome inhibitor treatment. This suggests a critical role of CHIP/CHN-1-mediated ubiquitylation in the control of muscle wasting and degeneration and identifies a potential new drug target for the treatment of this disease.

Topics: Actin Cytoskeleton; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Count; Cell Movement; Cell Nucleus; Genes, Helminth; Leupeptins; Muscle, Skeletal; Muscular Dystrophy, Animal; Mutation; Myosins; Phalloidine; Ubiquitin-Protein Ligases

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

Mdx mice have a genetic defect similar to that which causes Duchenne muscular dystrophy in humans. The influence of calcium on muscle protein metabolism of mdx and wild type (C57BL/10) mice was examined in vitro. Incubation of mdx muscles in a medium containing calcium at a concentration of 2.0 mM (but not 0.2 mM) resulted in proteolytic rates that were greater than those of C57BL/10 muscles. At 2.0 mM extracellular calcium, mdx muscle proteolysis was attenuated by thiol protease inhibitors but not by the weak base methylamine. Protein synthetic rates were higher in incubated mdx muscles than in incubated C57BL/10 muscles, but no effect of extracellular calcium concentration was observed in either strain. These data suggest that mdx mice have an abnormality of muscle calcium handling, which results in activation of nonlysosomal proteolytic processes but does not exert acute effects on protein synthetic rate.

Topics: Animals; Calcium; Cycloheximide; Kinetics; Leupeptins; Methylamines; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscles; Muscular Dystrophy, Animal; Proteins; Tyrosine

We compared the therapeutic effects of various low-molecular-weight enzyme inhibitors on dystrophic mice. Leupeptin, bestatin, forphenicinol and forphenicine significantly affected the enzymatic activities in the dystrophic muscles. The pattern of enzymatic changes in the muscles of forelimb and hindlimb caused by these inhibitors were similar in spite of the variety of their inhibitory spectra in vitro. However, comparing the pattern of enzymatic changes in spleen, forphenicinol differed from the other inhibitors tested. This may be related to the peculiar effects of this inhibitor on immunologically responsive cells.

Topics: Alkaline Phosphatase; Aminopeptidases; Animals; Esterases; Forelimb; Glutamyl Aminopeptidase; Glycine; Hindlimb; Leucine; Leucyl Aminopeptidase; Leupeptins; Male; Mice; Mice, Mutant Strains; Molecular Weight; Muscles; Muscular Dystrophy, Animal; Oligopeptides; Spleen

The activity of adenosine monophosphate (AMP)-aminohydrolase, the major NH3-producing enzyme in skeletal muscle, was approximately 35% lower in 7-day dystrophic muscle cell cultures than in normal muscle cell cultures. However, the release rate of NH3 from dystrophic muscle cells was 45% higher than that from normal muscle cells. The reasons for this apparent discrepancy are not clear. To determine indirectly if deamination of amino acids from protein degradation contributed to NH3 release, cells were incubated with 100 micrograms/ml of the protease inhibitor, leupeptin. Leupeptin reduced the rate of NH3 release by only 18.8% in normal muscle cells and 16% in dystrophic muscle cells. The release of NH3 was also higher from dystrophic chicken fibroblast cultures.

Topics: Adenosine Deaminase; Ammonia; Animals; Cells, Cultured; Chick Embryo; Fibroblasts; Leupeptins; Muscular Dystrophy, Animal; Protein Denaturation

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

For the purpose of observing the therapeutic benefit of protease inhibitors for progressive muscular dystrophy, a large quantity of doses of leupeptin of 10 mg/kg/day and 50 mg/kg/day were administered i.p. to male chickens afflicted with hereditary muscular dystrophy (line 413) for 4 months starting on the 7th day ex ovo. No clinical improvement was identified in physical ability as a result of the examination by flip test, and creatine kinase (CK) values. The number of necrotic fibers in the pectoralis superficialis (PS) muscle which is known to be preferentially damaged in dystrophic chicken, did not decrease significantly in the birds treated with 10 mg leupeptin/kg/day (number of necrotic fibers; 47.7/mm2) and 50 mg/kg/day (46.4/mm2) as compared to that of the untreated ones (43.2/mm2). A morphometric analysis of fiber diameter distribution also showed no statistical difference between the treated and untreated birds. In the second group, 10 mg leupeptin/kg and a combination of leupeptin and bestatin of 10 mg/kg each were injected directly into the left lower half of the PS muscle three times a week for 4 months. Necrotic fibers were still present in the injected site, remote area of the left upper PS muscle treated with leupeptin (52.7/mm2), leupeptin and bestatin (52.2/mm2), and contralateral right upper PS muscle (41.6 and 53.5/mm2, respectively). The number of necrotic fibers in treated muscles was again not significantly different from that in untreated dystrophic ones (39.6/mm2). In fiber diameter analysis, no statistical difference was recognized between the treated and untreated dystrophic muscles.

Topics: Aminopeptidases; Animals; Chickens; Creatine Kinase; Drug Therapy, Combination; Leucine; Leupeptins; Male; Muscles; Muscular Dystrophy, Animal; Necrosis; Oligopeptides

Mice with genetic muscular dystrophy were treated with intraperitoneal injections of the proteinase inhibitor leupeptin, beginning before the onset of weakness. A significant number of the treated animals failed to develop histological evidence of dystrophy, compared with controls. Leupeptin treatment prevented (or delayed) the onset of muscular dystrophy in this experiment.

Topics: Animals; Leupeptins; Mice; Muscular Dystrophy, Animal; Oligopeptides; Time Factors

Chicken myoblasts were cultured from the pectoralis muscles of dystrophic and normal 11-day-old embryos. Cells were allowed to grow to fusion (differentiation) and exposed to [35S]methionine for a short period. Subsequently, the decay of labeled proteins in the presence of cycloheximide was measured for various cellular fractions as well as individual proteins isolated from the sarcoplasmic reticulum and separated by gel electrophoresis. Some dystrophic material showed an increased decay when compared to normal material. The most significant (p less than 0.005) difference was found in a Mr = 65,000 component of the sarcoplasmic reticulum. This same component accumulates label at an accelerated rate in the presence of the protease inhibitor leupeptin. Increased turnover of this protein, possibly calsequestrin, may be a manifestation of the genetic disease.

Topics: Animals; Cells, Cultured; Chick Embryo; Cycloheximide; Kinetics; Leupeptins; Methionine; Muscle Proteins; Muscles; Muscular Dystrophy, Animal; Sarcoplasmic Reticulum

The total protein synthesis (TPS), myosin synthesis (MS) and creatine kinase (CK) levels in muscle cell cultures obtained from 400 normal (strain 454) and 400 dystrophic chick embryos (strain 455) were investigated. The cultures were obtained from breast muscles of 12 day chick embryos by dissociation in 0.25% trypsin, preplating and plating of 5 x 10(5) floating cells on gelatin coated dishes in Minimal Essential Medium, 10% horse serum and 2% chick embryo extract. After 6 days, when electron-microscopic studies demonstrated good muscle differentiation, cell cultures were labeled with [3H]leucine. TPS and MS, respectively, showed 85% and 65% increases in breast muscle cell cultures from dystrophic chick embryos. The half-life times for total protein and myosin from dystrophics were 19 and 32 hr, respectively as compared with 36 and 48 hr from controls. Noncollagen protein content (NCP) showed 27% decrease in postfusion stage (12 days) of cell cultures from dystrophics. The CK level showed 30% lower values in the cells from dystrophics but 50% higher values in their culture medium. The addition of leupeptin plus pepstatin (50 microgram/ml) to these cultures resotred NCP content, total protein and myosin turnover to normal values and significantly increased TPS and MS. The addition of diphenylhydantoin (DPH) (20 microgram/ml) to cell cultures from dystrophics did not change the NCP content nor the turnover for total protein and myosin but significantly increased TPS, MS and CK while medium CK significantly decreased. The addition of leupeptin plus pepstatin or DPH to muscle cell cultures from normal chick embryos also significantly stimulated TPS and MS.

Topics: Animals; Chick Embryo; Creatine Kinase; Culture Techniques; Leupeptins; Muscle Proteins; Muscles; Muscular Dystrophy, Animal; Myosins; Oligopeptides; Pepstatins; Phenytoin; Superoxide Dismutase

Topics: Animals; Cells, Cultured; Chickens; Leupeptins; Muscle Proteins; Muscles; Muscular Dystrophy, Animal; Oligopeptides

The protease inhibitor leupeptin decreases protein degradation in rat skeletal and cardiac muscle incubated in vitro, while protein synthesis remains unaltered. Leupeptin also lowers protein breakdown in denervated rat muscles and affected muscles from mice with hereditary muscular dystrophy. Leupeptin may thus be useful in retarding tissue atrophy. Since homogenates of leupeptin-treated muscles had decreased cathepsin B activity, this lysosomal protease may play a role in protein turnover in normal and diseased muscles.

Topics: Animals; Cathepsins; In Vitro Techniques; Leupeptins; Lysosomes; Muscle Denervation; Muscle Proteins; Muscles; Muscular Dystrophy, Animal; Myocardium; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Rats

The protease inhibitors leupeptin and pepstatin were used in vivo in genetically dystrophic chickens to determine their effects on the histological and biochemical changes observed in this disease. These compounds appear to delay the degeneration of muscle tissue which is characteristic of this disorder and thus may have potential therapeutic value in the treatment of muscular dystrophy.

Topics: Animals; Chickens; Creatine Kinase; Leupeptins; Muscles; Muscular Dystrophy, Animal; Oligopeptides; Pepstatins

Topics: Animals; Carbamates; Cells, Cultured; Chick Embryo; Drug Evaluation, Preclinical; Leupeptins; Muscles; Muscular Dystrophy, Animal; Oligopeptides; Pepstatins; Protease Inhibitors