Heat acclimation increases inflammatory and apoptotic responses to subsequent LPS challenge in C2C12 myotubes

This work investigated the ability of a 6-day heat acclimation protocol to impart heat acclimation-mediated cross-tolerance (HACT) in C2C12 myotubes, as indicated by changes in inflammatory and apoptotic responses to subsequent lipopolysaccharide (LPS) challenge. Myotubes were incubated at 40 °C for 2 h/day over 6 days (HA) or maintained for 6 days at 37 °C (C). Following 24 h recovery, myotubes from each group received either no stimulation or 500 ng/ml LPS for 2 h (HA + LPS and C + LPS, respectively). Cell lysates were collected and analyzed for protein markers of the heat shock response, inflammation, and apoptosis. As compared to C, HA exhibited an elevated heat shock response [HSP70 (+ 99%); HSP60 (+ 216%); HSP32 (+ 40%); all p < 0.01] and reduced inflammatory and apoptotic signaling [p-NF-ĸB:NF-ĸB (- 99%%); p-JNK (- 49%); all p < 0.01]. When compared to C + LPS, HA + LPS also exhibited an elevated heat shock response [HSP70 (+ 68%); HSP60 (+ 32%); HSP32 (+ 38%); all p < 0.01]. However, inflammatory and apoptotic responses in HA + LPS were increased [p-IKBa:IKBa (+ 432%); p-NF-ĸB:NF-ĸB (+ 283%); caspase-8p18 (+ 53%); p-JNK (+ 41%); all p < 0.05]. This unanticipated finding may be due to increased TLR4-mediated signaling capacity in HA + LPS, as indicated by upregulation of TLR4 [(+ 24%); MyD88 (+ 308%); p-NIK (+ 199%); and p-IKKα/b (+ 81%); all p < 0.05]. Data suggest HA reduces inflammatory and apoptotic signaling in skeletal muscle cells that are maintained under basal conditions. However, HACT is selective and does not apply to TLR4 signaling in the present model.