Neuromotor deficits and altered physiological responses to repeated exertional heat stroke exposures in mice.

Exertional heat stroke (EHS) is a life-threatening illness that can lead to negative health outcomes. Using a "severe" preclinical mouse model of EHS, we tested the hypotheses that one EHS exposure results in altered susceptibility to a subsequent EHS and reduced neuromotor performance. Female C57BL/6 mice underwent two protocols, 2 wk apart, either an EHS trial (EHS) or a sham exercise control trial (EXC). For EHS, mice ran in a forced running wheel at 37.5°C/40% relative humidity until loss of consciousness, followed by a slow cooling protocol (2 h recovery at 37.5°C). EXC mice exercised equally but in ∼22°C. Mice were randomized into three groups: 1) EXC-EXC (two consecutive EXC, n = 6, 2) EHS-EXC (EHS followed by EXC, n = 5), and 3) EHS-EHS (repeated EHS, n = 9). Mice underwent noninvasive neuromotor and behavioral tests during recovery and isolated soleus force measurements at the end of recovery. At the first EHS, mice reached average peak core temperatures (Tc,max) of 42.4°C, (46% mortality). On the second EHS, average Tc,max was reduced by ∼0.7°C (P < 0.05; mortality 18%). After the first EHS, both EHS-EX and EHS-EHS showed significant reductions in maximum strength (24 h and 1 wk post). After the second EHS, strength, horizontal rotation, hindlimb tone, suspended hindlimb splay, trunk curl, and provoked biting continued to decline in the EHS-EHS group. In conclusion, exposure to a second EHS after 2 wk leads to increased exercise times in the heat, symptom limitation at a lower Tc,max, and greater deficits in neuromotor and behavioral function during recovery.

Skeletal Muscle Interleukin-6 Contributes to the Innate Immune Response in Septic Mice.

ABSTRACT: Interleukin-6 (IL-6) is a major cytokine released by skeletal muscle. Although IL-6 plays complex but well-known roles in host defense, the specific contribution of skeletal muscle IL-6 to innate immunity remains unknown. We tested its functional relevance by exposing inducible skeletal muscle IL-6 knockdown (skmIL-6KD) mice to a cecal slurry model of polymicrobial peritonitis and compared responses to strain-matched controls and skeletal muscle Cre-matched controls at 3, 6, and 12 h postinfection. In both sexes, skmIL-6KD mice at 6 h of infection exhibited marked changes to leukocyte trafficking in the peritoneum, characterized by ∼1.75-fold elevation in %neutrophils, a ∼3-fold reduction in %lymphocytes and a ∼2 to 3-fold reduction in %basophils. A similar pattern was seen at 12 h. No changes were observed in plasma leukocyte counts. Circulating cytokines in female skmIL-6KD mice at 6 h consistently showed modest reductions in IL-6, but marked reductions in a broad range of both pro- and anti-inflammatory cytokines, e.g., TNFα and IL-10. In both sexes at 12 h, a generalized suppression of plasma cytokines was also seen after the effects of Cre-induction with raloxifene were addressed. There were no significant effects of skmIL-6KD on mortality in either sex. Collectively, our results are consistent with skmIL-6 playing an important and previously unrecognized role in immune cell trafficking and cytokine regulation during septic shock.

Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.

KEY POINTS: Exposure to exertional heat stroke (EHS) has been linked to increased long-term decrements of health. Epigenetic reprogramming is involved in the response to heat acclimation; however, whether the long-term effects of EHS are mediated by epigenetic reprogramming is unknown. In female mice, we observed DNA methylation reprogramming in bone marrow-derived (BMD) monocytes as early as 4 days of recovery from EHS and as late as 30 days compared with sham exercise controls. Whole blood, collected after 30 days of recovery from EHS, exhibited an immunosuppressive phenotype when challenged in vitro by lipopolysaccharide. After 30 days of recovery from EHS, BMD monocytes exhibited an altered in vitro heat shock response. The location of differentially methylated CpGs are predictive of both the immunosuppressive phenotype and altered heat shock responses. ABSTRACT: Exposure to exertional heat stroke (EHS) has been linked to increased susceptibility to a second heat stroke, infection and cardiovascular disease. Whether these clinical outcomes are mediated by an epigenetic memory is unknown. Using a preclinical mouse model of EHS, we investigated whether EHS exposure produces a lasting epigenetic memory in monocytes and whether there are phenotypic alterations that may be consistent with these epigenetic changes. Female mice underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation, characterized by CNS dysfunction. Results were compared with matched exercise controls at 22.5°C. Monocytes were isolated from bone marrow after 4 or 30 days of recovery to extract DNA and analyse methylation. Broad-ranging alterations to the DNA methylome were observed at both time points. At 30 days, very specific alterations were observed to the promoter regions of genes involved with immune responsiveness. To test whether these changes might be related to phenotype, whole blood at 30 days was challenged with lipopolysaccharide (LPS) to measure cytokine secretion; monocytes were also challenged with heat shock to quantify mRNA expression. Whole blood collected from EHS mice showed markedly attenuated inflammatory responses to LPS challenge. Furthermore, monocyte mRNA from EHS mice showed significantly altered responses to heat shock challenge. These results demonstrate that EHS leads to a unique DNA methylation pattern in monocytes and altered immune and heat shock responsiveness after 30 days. These data support the hypothesis that EHS exposure can induce long-term physiological changes that may be linked to altered epigenetic profiles.

Effects of Ibuprofen during Exertional Heat Stroke in Mice.

Intestinal injury is one of the most prominent features of organ damage in exertional heat stroke (EHS). However, whether damage to the intestine in this setting is exacerbated by ibuprofen (IBU), the most commonly used nonsteroidal anti-inflammatory drug in exercising populations, is not well understood. PURPOSE: We hypothesized that IBU would exacerbate intestinal injury, reduce exercise performance, and increase susceptibility to heat stroke. METHODS: To test this hypothesis, we administered IBU via diet to male and female C57/BL6J mice, over 48 h before EHS. Susceptibility to EHS was determined by assessing exercise response using a forced running wheel, housed inside an environmental chamber at 37.5°C. Core temperature (Tc) was monitored by telemetry. Mice were allocated into four groups: exercise only (EXC); EHS + IBU; EXC + IBU; and EHS only. Exercise performance and Tc profiles were evaluated and stomachs, intestines and plasma were collected at 3 h post-EHS. RESULTS: The EHS + IBU males ran approximately 87% longer when Tc was above 41°C (P < 0.03) and attained significantly higher peak Tc (P < 0.01) than EHS-only mice. Histological analyses showed decreased villi surface area throughout the small intestine for both sexes in the EXC + IBU group versus EXC only. Interestingly, though EHS in both sexes caused intestinal injury, in neither sex were there any additional effects of IBU. CONCLUSIONS: Our results suggest that in a preclinical mouse model of EHS, oral IBU at pharmacologically effective doses does not pose additional risks of heat stroke, does not reduce exercise performance, and does not contribute further to intestinal injury, though this could have been masked by significant gut injury induced by EHS alone.

Osmolality Selectively Offsets the Impact of Hyperthermia on Mouse Skeletal Muscle in vitro.

Hyperthermia and dehydration can occur during exercise in hot environments. Nevertheless, whether elevations in extracellular osmolality contributes to the increased skeletal muscle tension, sarcolemmal injury, and oxidative stress reported in warm climates remains unknown. We simulated osmotic and heat stress, in vitro, in mouse limb muscles with different fiber compositions. Extensor digitorum longus (EDL) and soleus (SOL) were dissected from 36 male C57BL6J and mounted at optimal length in tissue baths containing oxygenated buffer. Muscles were stimulated with non-fatiguing twitches for 30 min. Four experimental conditions were tested: isotonic-normothermia (285 mOsm•kg-1 and 35°C), hypertonic-normothermia (300 mOsm•kg-1 and 35°C), isotonic-hyperthermia (285 mOsm•kg-1 and 41°C), and hypertonic-hyperthermia (300 mOsm•kg-1 and 41°C). Passive tension was recorded continuously. The integrity of the sarcolemma was determined using a cell-impermeable fluorescent dye and immunoblots were used for detection of protein carbonyls. In EDL muscles, isotonic and hypertonic-hyperthermia increased resting tension (P < 0.001). Whereas isotonic-hyperthermia increased sarcolemmal injury in EDL (P < 0.001), this effect was absent in hypertonic-hyperthermia. Similarly, isotonic-hyperthermia elevated protein carbonyls (P = 0.018), a response not observed with hypertonic-hyperthermia. In SOL muscles, isotonic-hyperthermia also increases resting tension (P < 0.001); however, these effects were eliminated in hypertonic-hyperthermia. Unlike EDL, there were no effects of hyperthermia and/or hyperosmolality on sarcolemmal injury or protein carbonyls. Osmolality selectively modifies skeletal muscle response to hyperthermia in this model. Fast-glycolytic muscle appears particularly vulnerable to isotonic-hyperthermia, resulting in elevated muscle tension, sarcolemmal injury and protein oxidation; whereas slow-oxidative muscle exhibits increased tension but no injury or protein oxidation under the conditions and duration tested.