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The effect of exertional heat stroke (EHS) exposure on skeletal muscles is incompletely understood. Muscle weakness is an early symptom of EHS but is not considered a major target of multiorgan injury. Previously, in a preclinical mouse model of EHS, we observed the vulnerability of limb muscles to a second EHS exposure, suggesting hidden processes contributing to declines in muscle resilience. Here, we evaluated the possible molecular origins of EHS-induced declines in muscle resilience. Female C57BL/6 mice [total n = 56; 28/condition, i.e., EHS and exercise control (EXC)] underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation (unconsciousness). EXC mice exercised identically at room temperature (22-23°C). After 1 mo of recovery, the following were assessed: 1 ) specific force and caffeine-induced contracture in soleus (SOL) and extensor digitorum longus (EDL) muscles; 2 ) transcriptome and DNA methylome responses in gastrocnemius (GAST); and 3 ) primary satellite cell function (proliferation and differentiation). There were no differences in specific force in either SOL or EDL from EXC. Only EHS solei exhibited lower caffeine sensitivity. EHS GAST exhibited higher RNA expression of genes encoding structural proteins of slow fibers, heat shock proteins, and myogenesis. A total of ∼2,500 differentially methylated regions of DNA that could potentially affect many cell functions were identified. Primary satellite cells exhibited suppressed proliferation rates but normal differentiation responses. Results demonstrate long-term changes in skeletal muscles 1 mo after EHS that could contribute to declines in muscle resilience. Skeletal muscle may join other, more recognized tissues considered vulnerable to long-term effects of EHS. NEW & NOTEWORTHY Exertional heat stroke (EHS) in mice induces long-term molecular and functional changes in limb muscle that could reflect a loss of "resilience" to further stress. The phenotype was characterized by altered caffeine sensitivity and suppressed satellite cell proliferative potential. This was accompanied by changes in gene expression and DNA methylation consistent with ongoing muscle remodeling and stress adaptation. We propose that EHS may induce a prolonged vulnerability of skeletal muscle to further stress or injury.

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 (T c,max ) of 42.4°C, (46% mortality). On the second EHS, average T c,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 T c,max , and greater deficits in neuromotor and behavioral function during recovery.

Evidence from human epidemiological studies suggests that exertional heat stroke (EHS) results in an elevated risk of long-term cardiovascular and systemic disease. Previous results using a preclinical mouse model of EHS demonstrated severe metabolic imbalances in ventricular myocardium developing at 9-14 days of recovery. Whether this resolves over time is unknown. We hypothesized that the long-term effects of EHS on the heart reflect retained maladaptive epigenetic responses. In this study, we evaluated genome-wide DNA methylation, RNA-Seq, and metabolomic profiles of the left ventricular myocardium in female C57BL/6 mice, 30 days after EHS (exercise in 37.5°C; n = 7-8), compared with exercise controls. EHS mice ran to loss of consciousness, reaching core temperatures of 42.4 ± 0.2°C. All mice recovered quickly. After 30 days, the left ventricles were rapidly frozen for DNA methyl sequencing, RNA-Seq, and untargeted metabolomics. Ventricular DNA from EHS mice revealed >13,000 differentially methylated cytosines (DMCs) and >900 differentially methylated regions (DMRs; ≥5 DMCs with ≤300 bp between each CpG). Pathway analysis using DMRs revealed alterations in genes regulating basic cell functions, DNA binding, transcription, and metabolism. Metabolomics and mRNA expression revealed modest changes that are consistent with a return to homeostasis. Methylation status did not predict RNA expression or metabolic state at 30 days. We conclude that EHS induces a sustained DNA methylation memory lasting over 30 days of recovery, but ventricular gene expression and metabolism return to a relative homeostasis at rest. Such long-lasting alterations to the DNA methylation landscape could alter responsiveness to environmental or clinical challenges later in life.

New Findings: What is the topic of this review? This review outlines the history of research on epigenetic adaptations to heat exposure. The perspective taken is that adaptations reflect properties of hormesis, whereby low, repeated doses of heat induce adaptation (acclimation/acclimatization); whereas brief, life-threatening exposures can induce maladaptive responses. What advances does it highlight? The epigenetic mechanisms underlying acclimation/acclimatization comprise specific molecular programmes on histones that regulate heat shock proteins transcriptionally and protect the organism from subsequent heat exposures, even after long delays. The epigenetic signalling underlying maladaptive responses might rely, in part, on extensive changes in DNA methylation that are sustained over time and might contribute to later health challenges., Abstract: Epigenetics plays a strong role in molecular adaptations to heat by producing a molecular memory of past environmental exposures. Moderate heat, over long periods of time, induces an 'adaptive' epigenetic memory, resulting in a condition of 'resilience' to future heat exposures or cross-tolerance to other forms of toxic stress. In contrast, intense, life-threatening heat exposures, such as severe heat stroke, can result in a 'maladaptive' epigenetic memory that can place an organism at risk of later health complications. These cellular memories are coded by post-translational modifications of histones on the nucleosomes and/or by changes in DNA methylation. They operate by inducing changes in the level of gene transcription and therefore phenotype. The adaptive response to heat acclimation functions, in part, by facilitating transcription of essential heat shock proteins and exhibits a biphasic short programme (maintaining DNA integrity, followed by a long-term consolidation). The latter accelerates acclimation responses after de-acclimation. Although less studied, the maladaptive responses to heat stroke appear to be coded in long-lasting changes in DNA methylation near the promoter region of genes involved with basic cell function. Whether these memories are also encoded in histone modifications is not yet known. There is considerable evidence that both adaptive and maladaptive epigenetic responses to heat can be inherited, although most evidence comes from lower organisms. Future challenges include understanding the signalling mechanisms responsible and discovering new ways to promote adaptive responses while suppressing maladaptive responses to heat, as all life forms adapt to life on a warming planet., (© 2022 The Authors. Experimental Physiology © 2022 The Physiological Society.)

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., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

New Findings: What is the central question of this study? Exertional heat stroke is accompanied by a marked inflammatory response. In this study, we explored the time course of acute phase proteins during recovery from severe heat stress in mice and the potential role of skeletal muscles as their source. What is the main finding and its importance? Exertional heat stroke transiently increased expression of acute phase proteins in mouse liver and plasma and depleted liver and plasma fibrinogen, a typical response to severe trauma. In contrast, skeletal muscle fibrinogen production was stimulated by heat stroke, which can provide an additional reservoir for fibrinogen supply to maintain the clotting potential throughout the body and locally within the muscle., Abstract: Exertional heat stroke (EHS), the most severe manifestation of heat illness, is accompanied by a marked inflammatory response. The release of acute phase proteins (APPs) is an important component of inflammation, which can assist in tissue survival/repair. The time course of APPs in recovery from EHS is unknown. Furthermore, skeletal muscles produce APPs during infection, but it is unknown whether they can produce APPs after EHS. Our objective was to determine the time course of representative APPs in liver, plasma and skeletal muscle during recovery from EHS. Male C57BL6/J mice ran in a forced running wheel at 37.5°C, 40% relative humidity until symptom limitation. Exercise control (EXC) mice ran for the same duration and intensity at 22.5°C. Samples were collected (n = 6-12 per group) over 14 days of recovery. Protein abundance was quantified using immunoblots. Total and phosphorylated STAT3 (pSTAT3) at Tyr705, responsible for APP activation, increased in liver at 0.5 h after EHS compared with EXC, (P < 0.05 and P < 0.001, respectively). In contrast, in tibialis anterior (TA) muscle, total STAT3 increased at 3 h (P < 0.05) but pSTAT3 (Tyr705) did not. Liver serum amyloid A1 (SAA1) increased at 3 and 24 h after EHS (P < 0.05), whereas plasma SAA1 increased only at 3 h (P < 0.05). SAA1 was not detected in TA muscle. In liver and plasma, fibrinogen decreased at 3 h (P < 0.01) and increased in TA muscle (P < 0.05). Lipocalin-2 was undetectable in liver or TA muscle. Recovery from EHS is characterized by a transient acute phase response in both liver and skeletal muscle. However, APP expression profiles and subtypes differ between skeletal muscle and liver., (© 2020 The Authors. Experimental Physiology © 2020 The Physiological Society.)

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.

Exertional heat stroke (EHS) and malignant hyperthermia (MH) are life-threatening conditions, triggered by different environmental stimuli that share several clinical symptoms and pathophysiological features. EHS manifests during physical activity normally, but not always, in hot and humid environments. MH manifests during exposure to haloalkane anesthetics or succinylcholine, which leads to a rapid, unregulated release of calcium (Ca 2+ ) within the skeletal muscles inducing a positive-feedback loop within the excitation-contraction coupling mechanism that culminates in heat stroke-like symptoms, if not rapidly recognized and treated. Rare cases of awake MH, independent of anesthesia exposure, occur during exercise and heat stress. It has been suggested that EHS and MH are mediated by similar mechanisms, including mutations in Ca 2+ regulatory channels within the skeletal muscle. Rapid cooling, which is the most effective treatment for EHS, is ineffective as an MH treatment; rather, a ryanodine receptor antagonist drug, dantrolene sodium (DS), is administered to the victim to prevent further muscle contractions and hyperthermia. Whether DS can be an effective treatment for EHS victims remains uncertain. In the last decade, multiple reports have suggested a number of mechanistic links between EHS and MH. Here, we discuss aspects related to the pathophysiology, incidence, diagnosis and treatment. Furthermore, we present evidence regarding potential overlapping mechanisms between EHS and MH and explore current knowledge to establish what is supported by evidence or a lack thereof (i.e. conjecture).

Key Points: Exposure to exertional heat stroke (EHS) is associated with increased risk of long-term cardiovascular disorders in humans. We demonstrate that in female mice, severe EHS results in metabolic changes in the myocardium, emerging only after 9-14 days. This was not observed in males that were symptom-limited at much lower exercise levels and heat loads compared to females. At 14 days of recovery in females, there were marked elevations in myocardial free fatty acids, ceramides and diacylglycerols, consistent with development of underlying cardiac abnormalities. Glycolysis shifted towards the pentose phosphate and glycerol-3-phosphate dehydrogenase pathways. There was evidence for oxidative stress, tissue injury and microscopic interstitial inflammation. The tricarboxylic acid cycle and nucleic acid metabolism pathways were also negatively affected. We conclude that exposure to EHS in female mice has the capacity to cause delayed metabolic disorders in the heart that could influence long-term health., Abstract: Exposure to exertional heat stroke (EHS) is associated with a higher risk of long-term cardiovascular disease in humans. Whether this is a cause-and-effect relationship remains unknown. We studied the potential of EHS to contribute to the development of a 'silent' form of cardiovascular disease using a preclinical mouse model of EHS. Plasma and ventricular myocardial samples were collected over 14 days of recovery. Male and female C57bl/6J mice underwent forced wheel running for 1.5-3 h in a 37.5°C/40% relative humidity until symptom limitation, characterized by CNS dysfunction. They reached peak core temperatures of 42.2 ± 0.3°C. Females ran ∼40% longer, reaching ∼51% greater heat load. Myocardial and plasma samples (n = 8 per group) were obtained between 30 min and 14 days of recovery, analysed using metabolomics/lipidomics platforms and compared to exercise controls. The immediate recovery period revealed an acute energy substrate crisis from which both sexes recovered within 24 h. However, at 9-14 days, the myocardium of female mice developed marked elevations in free fatty acids, ceramides and diacylglycerols. Glycolytic and tricarboxylic acid cycle metabolites revealed bottlenecks in substrate flow, with build-up of intermediate metabolites consistent with oxidative stress and damage. Males exhibited only late stage reductions in acylcarnitines and elevations in acetylcarnitine. Histopathology at 14 days showed interstitial inflammation in the female hearts only. The results demonstrate that the myocardium of female mice is vulnerable to a slowly emerging metabolic disorder following EHS that may harbinger long-term cardiovascular complications. Lack of similar findings in males may reflect their lower heat exposure., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)