Interaction of Hyperthermia and Ligand‐Mediated IL‐6 Production in Mouse Skeletal Muscle

Skeletal muscles produce cytokines including interleukin‐6 (IL‐6) in response to perturbations from homeostasis. Exposure to hyperthermia, epinephrine (EPI), and lipopolysaccharide (LPS) alone all result in increased skeletal muscle IL‐6 production. Combined presence of hyperthermia with circulating EPI or LPS ligands is found in conditions of fatiguing exercise and bacterial septicemia. We hypothesize that hyperthermia will amplify IL‐6 production by skeletal muscle in response to EPI or LPS. Mouse solei were both excised in oxygenated Krebs Ringer solution and placed under 1 gram of resting tension in temperature controlled baths. Solei from each mouse were matched, one treated at 35°C and one at 41°C for 1 hour, both followed by 1 hour at 35°C. Muscles were exposed in the bath to either low dose (1ng/ml) or high dose (100ng/ml) EPI, or low dose (.0002μg/ml) or high dose (1μg/ml) LPS. Bath samples at hour 1 (T1) and 2 (T2) were collected, mixed with antiprotease, flash frozen in liquid nitrogen, and stored at −80°C prior to Luminex multiplex analysis. At T2, solei were blotted on tissue paper, flash frozen, and stored for mRNA analyses. mRNA responses were expressed as a fold change relative to a naïve control soleus muscle. Effects of heat were tested for significant differences in mRNA and protein via Wilcoxin signed ranks for paired measurements. Effect of LPS or EPI alone on protein excretion were compared to buffer via Wilcoxin signed ranks test. IL‐6 gene expression increased in response to 41°C at low (4.62‐fold; p=.031) and high (9.57‐fold; p=.031) doses of EPI, and high dose LPS (2.05‐fold; p=.031). High dose EPI and LPS increased IL‐6 protein excretion (p=0.016, T1 & T2), but with no additional effect of heat. Hyperthermia coinciding with IL‐6 stimulating ligands (EPI & LPS) increases mRNA above ligand alone, without subsequent additional increases in protein excretion. Support or Funding Information The work was supported by American Heart Association #11GRNT7990119 (TLC) and University of Florida BK and Betty Stevens Endowment (TLC). 1 Interaction of hyperthermia and high dose EPI (100ng/ml) or LPS (1μg/ml) stimulated IL‐6 regulation in isolated soleus muscleA) Significant interaction of heat and EPI/LPS on fold change increase in IL‐6 mRNA at 2 h relative to naïve control (Wilcoxin signed ranks for paired measures). No significant interaction of heat and B) EPI or C) LPS on IL‐6 protein secretion rate; significant elevations in secreted IL‐6 above T0 at 1 and 2 h in all groups (Wilcoxin signed ranks).2 Low dose EPI (1ng/ml) or LPS (.0002μg/ml) replication of Figure 1 experimentA) Significant interaction of heat and EPI on fold change increase in IL‐6 mRNA at 2 h relative to naïve control (Wilcoxin signed ranks for paired measures). No significant interaction of heat and B) EPI or C) LPS on IL‐6 protein secretion rate; significant elevations in secreted IL‐6 above T0 at 1 and 2 h in heat treated groups; and 1 h EPI and 2 h LPS normothermic groups (Wilcoxin signed ranks).