Inhibition of iPLA2β and of stretch-activated channels by doxorubicin alters dystrophic muscle function

BACKGROUND AND PURPOSE Chronic elevation in intracellular Ca2+ concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca2+ overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca2+-independent form of PL A(2) (iPLA(2)). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA(2) in other systems, on the activity of this enzyme and on the consequences on Ca2+ handling and muscle function in mdx mice. EXPERIMENTAL APPROACH Effects of Dox on iPLA(2) activity, reactive oxygen species production and on Ca2+ influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA(2) inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca2+ 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 mM inhibited iPLA(2) activity in cells and in the purified enzyme. Dox also inhibited SAC-but not SOC-mediated Ca2+ influx in myotubes. Stimulated elevations of Ca2+ 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 iPLA(2) and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.