Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction

Calpain activation contributes to the development of infection-induced diaphragm weakness, but the mechanisms by which infections activate calpain are poorly understood. We postulated that skeletal muscle calcium-dependent phospholipase A(2) (cPLA(2)) is activated by cytokines and has downstream effects that induce calpain activation and muscle weakness. We determined whether cPLA(2) activation mediates cytokine-induced calpain activation in isolated skeletal muscle (C2C12) cells and infection-induced diaphragm weakness in mice. C2C12 cells were treated with the following: 1) vehicle; 2) cytomix (TNF-alpha 20 ng/ml, IL-1 beta 50 U/ml, IFN-gamma 100 U/ml, LPS 10 mu g/ml); 3) cytomix + AACOCF3, a cPLA(2) inhibitor (10 mu M); or 4) AACOCF3 alone. At 24 h, we assessed cell cPLA(2) activity, mitochondrial superoxide generation, calpain activity, and calpastatin activity. We also determined if SS31 (10 mu g/ml), a mitochondrial superoxide scavenger, reduced cytomix-mediated calpain activation. Finally, we determined if CDIBA (10 mu M), a cPLA(2) inhibitor, reduced diaphragm dysfunction due to cecal ligation puncture in mice. Cytomix increased C2C12 cell cPLA(2) activity (P < 0.001) and superoxide generation; AACOCF3 and SS31 blocked increases in superoxide generation (P < 0.001). Cytomix also activated calpain (P < 0.001) and inactivated calpastatin (P < 0.01); both AACOCF3 and SS31 prevented these changes. Cecal ligation puncture reduced diaphragm force in mice, and CDIBA prevented this reduction (P < 0.001). cPLA(2) modulates cytokine-induced calpain activation in cells and infection-induced diaphragm weakness in animals. We speculate that therapies that inhibit cPLA(2) may prevent diaphragm weakness in infected, critically ill patients.