ABSTRACT
BACKGROUND AND PURPOSE: Chronic elevation in intracellular Ca(2+) concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca(2+) overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca(2+) -independent form of PL A2 (iPLA2 ). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca(2+) handling and muscle function in mdx mice. EXPERIMENTAL APPROACH: Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca(2+) influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca(2+) concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. KEY RESULTS: Dox at 1-30 µM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca(2+) influx in myotubes. Stimulated elevations of Ca(2+) concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. CONCLUSIONS AND IMPLICATIONS: Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.
Subject(s)
Calcium Channels/metabolism , Doxorubicin/pharmacology , Group VI Phospholipases A2/antagonists & inhibitors , Muscle Contraction/drug effects , Muscle Fibers, Skeletal/physiology , Reactive Oxygen Species/metabolism , Animals , Calcium Signaling/drug effects , Cell Line , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle Contraction/physiology , Muscle Fibers, Skeletal/pathologyABSTRACT
BACKGROUND AND PURPOSE: Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by the absence of the cytoskeletal protein dystrophin. This leads to muscle cell death accompanied by chronic inflammation. Cyclosporin A (CsA) is a powerful immunosuppressive drug, which has been proposed for DMD treatment. CsA also directly regulates the mitochondrial permeability transition pore (mPTP), which participates in cell death pathways through the inhibition of cyclophilin D. Here, we evaluated whether Debio 025, a cyclophilin inhibitor with no immunosuppressive activity, improves the dystrophic condition in a mouse model of DMD, through regulation of mPTP. EXPERIMENTAL APPROACH: The potency of Debio 025 to protect mouse dystrophic cells against mitochondria-mediated death was assessed by caspase-3 activity and calcium retention capacity assays. Mdx(5Cv) mice (3-week-old) were treated daily by gavage for 2 weeks with Debio 025 (10, 30 or 100 mg kg(-1)), CsA (10 mg kg(-1)) or placebo. The effects on muscle necrosis and function were measured. KEY RESULTS: In vitro investigations showed protective effect of low concentrations of Debio 025 against cell death. Histology demonstrated that Debio 025 partially protected the diaphragm and soleus muscles against necrosis (10 and 100 mg kg(-1), respectively). Hindlimb muscles from mice receiving Debio 025 at 10 mg kg(-1) relaxed faster, showed alteration in the stimulation frequency-dependent recruitment of muscle fibres and displayed a higher resistance to mechanical stress. CONCLUSIONS AND IMPLICATIONS: Debio 025 partially improved the structure and the function of the dystrophic mouse muscle, suggesting that therapies targeting the mPTP may be helpful to DMD patients.
Subject(s)
Cyclophilins/antagonists & inhibitors , Cyclosporine/pharmacology , Muscular Dystrophy, Animal/drug therapy , Muscular Dystrophy, Duchenne/drug therapy , Animals , Animals, Newborn , Cell Death/drug effects , Cyclophilins/metabolism , Cyclosporine/administration & dosage , Diaphragm/metabolism , Diaphragm/physiopathology , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Delivery Systems , Female , Hindlimb , Male , Mice , Mice, Inbred mdx , Mitochondrial Membrane Transport Proteins/drug effects , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiopathology , Necrosis/drug therapy , Necrosis/etiologyABSTRACT
To study the pathogenesis of X-linked recessive myotubular myopathy (XLMTM), we used a nerve-muscle coculture system which allows the reconstitution of functional motor units in vitro after coupling of human skeletal muscle cells with embryonic rat spinal cord explants. We used three skeletal muscle cell lines derived from subjects with known mutations in the MTM1 gene (two from embryonic tissues, associated with mutations predicted to give a severe phenotype, and one from a neonate still alive at 3 years 6 months and exhibiting a mild phenotype). We compared these three XLMTM muscle cell cultures with control cultures giving special attention to behaviour of living cocultures (formation of the myofibres, contractile activity, survival), expression of muscular markers (desmin, dystrophin, alpha-actinin, troponin-T, myosin heavy chain isoforms), and nerve-muscle interactions (expression and aggregation of the nicotinic acetylcholine receptors). We were unable to reproduce any 'myotubular' phenotype since XLMTM muscle cells behaved like normal cells with regard to all the investigated parameters. Our results suggest that XLMTM muscle might be intrinsically normal and emphasize the possible involvement of the myotubularin-deficient motor neurons in the development of the disease.
