Post-exertional heat stroke inhibition of c-Jun NH ₂ -terminal Kinase neither improves symptoms nor accelerates recovery

Organisms develop heat tolerance following short-term exposure to moderate heating, being able to withstand subsequent extreme heat that would otherwise be lethal. The phenomenon is conserved across taxa, but its efficacy against the most severe heat illness, exertional heat stroke (EHS), has not been tested. Previous data also indicate phosphorylation of c-Jun NH 2 -terminal kinase (JNK) in several organs corresponds to heat illness severity. Accordingly, there were 2 aims for the current study: 1) determine whether heat tolerance following a single heat preconditioning bout improves EHS symptoms & outcomes and 2) elucidate the role of JNK during heat illness (i.e., could its inhibition underlie heat tolerance mechanisms?). We subjected mice to EHS following 1 bout of preconditioning: 1) heat+exercise (HEx-EHS), 2) normothermal exercise (Ex-EHS), or 3) sedentary (Sed-EHS). An Exercise Control group (Sed-ExC) was also included for non-pathological, exertional conditions. For preconditioning, all mice were placed in a forced-running wheel. For Sed preconditioning, wheels were stationary and locked; for HEx and Ex preconditioning, wheels gradually increased from low-to-moderate speed. Ambient temperature was set at 25°C for Sed and Ex preconditioning and 37.5°C for HEx preconditioning (humidity at 30-70% for all). Precondition durations were time-matched to HEx specimens, which ran until core body temperature (T C ) was between 41.0-41.5°C for 20 minutes; running was immediately halted if T C reached 41.5°C prior to 20 minutes. Following 24h of recovery in their home cages at 25°C, mice were subjected to EHS by running in 37.5°C conditions until symptom limitation. HEx-EHS mice (256±35 min) had delayed EHS onset compared to Ex-EHS (228±28 min; p<0.001) and Sed-EHS (202±22 min; p<0.001). Additionally, no fatalities were observed in the HEx-EHS mice, but ~7-10% mortality were observed in the other EHS groups. To inhibit JNK phosphorylation, SP600125 or vehicle was injected immediately post-EHS in a separate cohort of EHS mice without preconditioning. Compared to vehicle, SP600125 did not accelerate EHS recovery nor improve outcomes. Preliminary western blot analysis of livers also indicated that SP600125 did not alter JNK phosphorylation, but there was a trend for inhibition of c-Jun phosphorylation on Serine 73 compared to vehicle (n=5-6/group; p = 0.077). We provide proof of concept that a single bout of heat preconditioning in mice delays EHS onset and mitigates its symptoms compared to exercise or sedentary preconditions. We have also determined that although JNK phosphorylation positively correlates to heat illness severity, its inhibition does not mitigate EHS symptoms and may be protective during heat illness. Disclaimer: The opinions or assertions contained herein are the private views of the author(s) and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations. No conflicts of interest, financial or otherwise, are declared by the authors. Military Operational Medicine Research Program: MO230069 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.