Free Radical–Induced Contractile Protein Dysfunction in Endotoxin-Induced Sepsis

Abstract

Recent studies have indicated that sepsis is associated with enhanced generation of several free-radical species (nitric oxide [NO], superoxide, hydrogen peroxide) in skeletal muscle. It is also known that this enhanced free-radical generation results in reductions in skeletal muscle force-generating capacity, but the precise mechanism(s) by which free radicals exert this effect in sepsis has not been determined. We postulated that free radicals might react directly with the contractile proteins in this condition, altering contractile protein force-generating capacity. To test this theory, we compared the force generation of single Triton-skinned diaphragmatic fibers (Triton skinning exposes the contractile apparatus, permitting direct assessment of contractile protein function) from the following groups of rats: (1) control animals; (2) endotoxin-treated animal; (3) animals given endotoxin plus polyethylene glycol- superoxide dismutase (PEG-SOD), a superoxide scavenger; (4) animals given endotoxin plus N(omega)-nitro-L-arginine methylester (L-NAME), a NO synthase inhibitor; (5 ) animals given only PEG-SOD or L-NAME; and (6 ) animals given endotoxin plus denatured PEG-SOD. We found that endotoxin administration produced both a reduction in the maximum force-generating capacity (Fmax) (i.e., a decrease in Fmax) of muscle fibers and a reduction in fiber calcium sensitivity (i.e., an increase in the Ca2+ concentration required to produce half-maximal activation [Ca50]). L-NAME and PEG-SOD administration preserved Fmax and Ca50 in endotoxin-treated animals; neither drug affected these parameters in non-endotoxin treated animals. Denatured PEG-SOD failed to inhibit endotoxin-related alterations in contractile protein function. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of skinned fibers from endotoxin-treated animals revealed a selective depletion of several proteins; administration of L-NAME or PEG-SOD to endotoxin-treated animals prevented this protein depletion, paralleling the effect of these two agents to prevent a reduction in contractile protein force-generating capacity. These data indicate that free radicals (superoxide, NO, or daughter species of these radicals) play a central role in altering skeletal muscle contractile protein force-generating capacity in endotoxin-induced sepsis.

Nuclear factor-kappa B signaling in skeletal muscle atrophy

“…Because sepsis is an inflammatory disorder, increased levels of cytokines and chemokines might play a prominent role in skeletal muscle-wasting during sepsis [77]. Infiltration of macrophages and neutrophils has been strongly implicated in the pathogenesis of sepsis-induced diaphragmatic weakness [78]. The chemokines of the CC and CXC family, which are involved in the recruitment of macrophages and neutrophils, are highly expressed in the septic mouse diaphragm in vivo.…”

Calpain Activation Contributes to Endotoxin-Induced Diaphragmatic Dysfunction

“…Previous work has demonstrated that administration of PEG-SOD, a potent superoxide scavenger, also attenuates endotoxininduced diaphragm dysfunction (12). To determine if a potential link exists between superoxide generation and calpain activation, we examined the effect of administration of PEG-SOD on endotoxin-induced diaphragmatic calpain activation in mice.…”

“…Moreover, it is known that endotoxin administration results in a significant increase in indices of oxidative stress in the diaphragm (12). Taking these two facts together, it seemed reasonable to ask whether or not calpain activation in the diaphragm in sepsis was linked, in some way, to levels of oxidative stress.…”

p38 Mitogen-Activated Protein Kinase Modulates Endotoxin-Induced Diaphragm Caspase Activation

“…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)(5)(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).…”