Sepsis increases contraction-related generation of reactive oxygen speciesin the diaphragm

Recent work indicates that free radicals mediate sepsis-induced diaphragmat ic dysfunction. These previous experiments have not, however, established t he source of the responsible free radical species. In theory, this phenomen on could be explained if one postulates that sepsis elicits an upregulation of contraction-related free radical formation in muscle. The purpose of th e present study was to test this hypothesis by examination of the effect of sepsis on contraction-related free radical generation [i.e., formation of reactive oxygen species (ROS)] by the diaphragm. Rats were killed 18 h afte r injection with either saline or endotoxin. In vitro hemidiaphragms were t hen prepared, and ROS generation during electrically induced contractions ( 20-Hz trains delivered for 10 min) was assessed by measurement of the conve rsion of hydroethidine to ethidium. ROS generation was negligible in noncon tracting diaphragms from both saline-and endotoxin-treated groups (2.0 +/- 0.6 and 2.8 +/- 1.0 ng ethidium/mg tissue, respectively), but it was marked in contracting diaphragms from saline-treated animals (19.0 +/- 2.8 ng/mg tissue) and even more pronounced (30.0 +/- 2.8 ng/mg tissue) in diaphragms from septic animals (P < 0.01). This enhanced free radical generation occur red despite the fact that the force-time integral(i.e., the area under the curve of force vs. time) for control diaphragms was higher than that for th e septic group. In additional studies, in which we altered the stimulation paradigm in control muscles to achieve a force-time integral similar to tha t achieved in septic muscles, an even greater difference between control an d septic muscle ROS formation was observed. These data indicate that ROS fo rmation during contraction is markedly enhanced in diaphragms from endotoxi n-treated septic animals. We speculate that ROS generated in this fashion p lays a central role in producing sepsis-related skeletal muscle dysfunction .