Optimization of a Rhabdomyolysis Model in Mice With Exertional Heat Stroke Mouse Model of EHS-Rhabdomyolysis

Abstract

Exertional heat stroke (EHS) is a life-threatening disease characterized by high mortality and incidence of rhabdomyolysis (RM). It would therefore be valuable to establish a stable EHS-induced RM model that accurately reflects the clinical characteristics of EHS patients and provides an objective animal model for further study of the pathogenesis of RM. In the current study, 8∼9-week-old, male, wild-type C57BL/6J mice, at the stage of sexual maturity, were randomly divided into four groups: the EHS group, the classical heat stroke (CHS) group, the sham heat exercise group, and sham heat rest group. The survival rate of mice was determined under relatively high levels of temperature and humidity (37.5 • C, 65% relative humidity (RH); 37.5 • C, 70% RH; 39.5 • C, 65% RH; and 39.5 • C, 70% RH) as well as a high core temperature (Tc; 42, 42.5, and 43 • C). Results showed that the environmental condition of 39.5 • C and 65% RH was most suitable for EHS modeling. The end point of EHS evaluation was exhaustion or an individual's core temperature reaching 43 • C. The survival rate of mice in the EHS group within 24 h under these conditions was 37.34%, which is consistent with the high mortality characteristics noted in EHS patients. Severe RM was observed in the EHS group by H&E staining and transmission electron microscopy. Creatine kinase levels in the EHS group mostly exceeded 10,000 U/L, which was approximately 10 times higher than that in the sham heat rest group. Renal tubules of the EHS group exhibited severe necrosis, and calcium overload in the skeletal muscles of this group was also observed using intravital 2photon microscopy. In conclusion, we made improvements to a stable EHS-induced RM animal model to truly reflect the clinical characteristics of EHS patients. This new model should be helpful in the further study of RM pathogenesis.

ACSL4 contributes to ferroptosis‐mediated rhabdomyolysis in exertional heat stroke

“… 10 The mechanism by which EHS induces RM remains unclear. In the current study, using a murine EHS model, 18 we uncovered an important role of ACSL4 in mediating EHS‐induced RM. We demonstrated that EHS‐induced YAP acting through TEAD1/TEAD4 upregulates ACSL4, which, in turn, promotes lipid peroxidation, skeletal muscle cells ferroptosis, thereby, leading to loss of functional skeletal muscle cells and subsequent RM ( Figure 7 ).…”

“…In this study, using the previous established murine EHS model, 18 we demonstrate that YAP, acting through TEAD1/TEAD4 relevant Hippo signalling, mediated EHS‐induced upregulation of ACSL4 which in turn, increased lipid peroxiadation level, as well as skeletal muscle cell ferroptosis and subsequent augmented skeletal muscle tissue injury. Inhibition of YAP, pharmacological inhibition of ACSL4, or blocking lipid peroxidation prevented EHS‐induced ferroptosis and ameliorated skeletal muscle tissue injury.…”

“…Our previous study also demonstrated that the mortality rate of EHS model with RM could reached to 62.66% within 24 h after EHS. 18 To evaluate the therapeutic effects of ferroptosis activation on the mortality rate of EHS mice within 24 h, we administered the single dose of Fer‐1 at 0, 6, and 12 h to the mice separately after EHS and then measured the mortality rate within 24 h after EHS, respectively. Our analysis revealed that the single‐dose treatment of Fer‐1 at 0 h powerfully improved the survival rate from EHS within 24 h and remarkably blocked skeletal muscle injury induced by EHS, as indicated by the decrease in CK and MB levels at 6 h after EHS ( Figure S5A – S5C ).…”

“…To investigate the activation of ferroptosis in RM in vitro , the primary myoblasts cells were exposed to ionomycin plus heat stress (io + hs) for simulating the effects of exercise and heat stress. 18 For the treatment of ionomycin leads to calcium overload, which is frequently accompanied by RM following EHS. 20 The treatment of Fer‐1 protect against the primary myoblasts death induced by io + hs, as shown by the decrease in LDH release ( Figure 2H ); the inhibited lipid peroxidation, as indicated by C11 BODIPY 581/591 fluorescence ( Figure 2I ); and the reduced lipid metabolites levels of MDA, 5‐HETE, and 15‐HETE ( Figure 2J–2L ).…”

“…Immediately after the onset of EHS, the mice were then removed from the climate chamber, weighed, and returned to their original cages with an ambient temperature of 25°C and with ad libitum access to food and water. 18 …”