Complement activation alters myocellular sodium homeostasis during polymicrobial sepsis

Objective: To determine whether complement activation alters sodium homeostasis in fast-twitch skeletal muscles during sepsis, and if protein kinase-C is involved in this process. Design: Prospective, randomized, controlled animal study. Setting: Research laboratory. Subjects: Male Sprague-Dawley rats weighing 60-75 g. Interventions: Rats underwent cecal ligation and puncture (CLP) or sham-operation with or without soluble complement receptor-1 treatment. Soluble complement receptor-1 (20 mg/kg) was administered intraperitoneally 5 mins before operation. Twenty-four hours after operation, fast-twitch extensor digitorum longus muscles were isolated and incubated in normal Krebs-Henseleit buffer (pH 7.4). In addition, extensor digitorum longus muscles isolated from normal rats were incubated for 1 hr in the Krebs-Henseleit buffer media containing normal rat sera, zymosan-activated (4 or 10 mg/mL) rat sera, or heat-inactivated rat sera. Ten percent diluted rat sera were used as a complement source in all groups. Last, extensor digitorum longus muscles isolated from normal rats were incubated for 1 hr in the Krebs-Henseleit buffer media containing zymosan-activated or heat-inactivated rat sera in the presence of protein kinase-C inhibitors (i.e., 4 μM GF109203X or 5 μM rottlerin). Soluble C5b-9 complex concentrations in zymosan-activated human sera were determined by enzyme-linked immunosorbent assay to evaluate the degree of complement activation induced by zymosan. Measurements and Main Results: Incubated extensor digitorum longus muscles from CLP, sham-operated, or normal rats were used to measure intracellular Na⁺ and K⁺ contents ([Na⁺]_i or [K⁺]_i). Polymicrobial sepsis, as produced by CLP, markedly increased [Na⁺]_i and [Na⁺]_i/[K⁺]_i ratios in fast-twitch extensor digitorum longus muscles 24 hrs after CLP compared with sham operation. Administration of soluble recombinant complement receptor 1 before operation significantly decreased myocellular [Na⁺]_i and [Na⁺]_i/[K⁺]_i ratios. Zymosan profoundly elevated soluble C5b-9 concentrations in human sera in vitro. Sublytic zymosan-activated rat sera significantly increased myocellular [Na⁺]_i and [Na⁺]_i/[K⁺]_i ratios relative to heat-inactivated rat sera. No difference in myocellular [Na⁺]_i and [Na⁺]_i/[K⁺]_i ratios was observed when we used 4 mg/mL compared with 10 mg/mL of zymosan for activation. Last, incubation of extensor digitorum longus muscles with GF109203X or rottlerin significantly attenuated increases in myocellular [Na⁺]_i and [Na⁺]_i/[K⁺]_i ratios induced by sublytic zymosan-activated rat sera. Conclusions: Polymicrobial sepsis alters sodium homeostasis in fast-twitch skeletal muscles, which is significantly attenuated by administration of soluble complement receptor 1. Protein kinase-C inhibition completely blocks changes in myocellular [Na⁺]_i and [Na⁺]_i/[K⁺]_i ratios induced by sublytic zymosan-activated rat sera. Collectively, these results suggest that an inappropriate activation of complement is, at least in part, responsible for changes in skeletal muscle sodium homeostasis during sepsis, and activation of PKC is one of the intracellular signaling pathways by which complement activation alters myocellular sodium homeostasis.