Double-stranded RNA-dependent protein kinase activation modulates endotoxin-induced diaphragm weakness

Diaphragm caspase-8 activation plays a key role in modulating sepsis-induced respiratory muscle dysfunction. It is also known that double-stranded RNA-dependent protein kinase (PKR) is a regulator of caspase-8 activation in neural tissue. We tested the hypothesis that the PKR pathway modulates sepsis-induced diaphragmatic caspase-8 activation. We first evaluated the time course of diaphragm PKR activation following endotoxin administration in mice. We then determined whether administration of a PKR inhibitor (2-aminopurine) prevents endotoxin-induced diaphragm caspase-8 activation and contractile dysfunction in mice. Finally, we investigated if inhibition of PKR (using either 2-aminopurine or transfection with dominant-negative PKR) blocks caspase-8 activation in cytokine treated C₂C₁₂ cells. Endotoxin markedly activated diaphragm PKR (with increases in both active phospho-PKR protein levels, P < 0.03, and directly measured PKR activity, P < 0.01) and increased active caspase-8 levels (P < 0.01). Inhibition of PKR with 2-aminopurine prevented endotoxininduced diaphragm caspase-8 activation (P < 0.01) and diaphragm weakness (P < 0.001). Inhibition of PKR with either 2-aminopurine or transfection with dominant-negative PKR blocked caspase-8 activation in isolated cytokine-treated C₂C₁₂ cells. These data implicate PKR activation as a major factor mediating cytokine-induced skeletal muscle caspase-8 activation and weakness.