We postulated that the p38 pathway is activated in the diaphragm during sepsis and contributes to sepsis-induced diaphragm caspase activation and contractile dysfunction. This study determined whether: (1) endotoxin administration elicits p38 activation in the diaphragm; (2) cytokines activate p38 in isolated muscle cells; (3) activation of p38 is accompanied by caspase 8 activation; (4) inhibition of p38 prevents caspase 8 activation and; (5) inhibition of p38 prevents diaphragm dysfunction in endotoxin-treated animals. We first evaluated the time course of diaphragm p38 activation after endotoxin in mice. We then determined if p38 inhibitor administration could prevent caspase 8 activation in endotoxin-treated mice. We also assessed p38 and caspase 8 activation in C2C12 muscle cells treated with control media or a cytokine mixture, with or without concomitant chemical inhibition of p38 (using SB203580, 25 mM) or loss of p38 function due to cell transfection with a dominant negative p38 genetic construct. Endotoxin administration activated diaphragm p38 (P , 0.001), and cytokines activated p38 in C2C12 cells (P , 0.05). In both the diaphragm and cells, p38 activation was accompanied by increases in active caspase 8 (P , 0.01). Inhibition of p38 with either SB203580 or with a dominant negative p38 construct prevented caspase activation (P , 0.001). p38 inhibitors also prevented endotoxin-induced diaphragm weakness (P , 0.001). p38 modulates cytokine-induced skeletal muscle caspase activation.Keywords: diaphragm; endotoxin; caspase; proteolysis; sepsis Two recent studies performed with the magnetic twitch stimulation technique found that critically ill patients develop severe diaphragm weakness, averaging diaphragm twitch forces that are only 20-25% of those observed in normal subjects (1, 2). This severe weakness is thought to account, in part, for the difficulty weaning many patients in intensive care units (ICUs) from mechanical ventilation (3). The mechanisms responsible for this severe respiratory muscle weakness in patients in the ICU are poorly understood, but many of these patients have infections, and infections have been repeatedly shown to induce severe respiratory muscle weakness (4-6). In recent studies, we have shown that endotoxin-induced sepsis potently activates diaphragmatic caspase 8, and that caspase 8 activation is linked, in turn, to the development of diaphragmatic weakness (7,8). It is also known that infections strongly activate the mitogenactivated protein kinase (MAPK) pathways in many tissues, and that the p38 MAPK pathway can elicit caspase activation and cellular dysfunction in a number of organs (9-11). However, the role of the p38 kinase pathway in modulating infection-induced diaphragm muscle caspase activation has never been studied.The purpose of the present group of experiments, therefore, was to test the hypothesis that endotoxin-induced sepsis leads to p38 pathway activation and p38-mediated activation of diaphragmatic caspase 8. In whole-animal studies, sepsis was induced by...