Inhibition of iPLA2 β and of stretch-activated channels by doxorubicin alters dystrophic muscle function.

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.