Changes in DNA Methylation Following Exertional Heat Exhaustion, Heat Injury, or Heat Stroke in Humans

Background: DNA methylation has emerged as a potential epigenetic mechanism influencing adaptations to heat illness in mice, yet methylation patterns that occur with heat illness in humans is unknown. Purpose: The purpose of this study was to assess methylation patterns induced by heat exhaustion, heat injury, or exertional heat stroke (EHS) in humans, representing increasing severity of heat illness, respectively. Hypothesis: Our hypothesis was that methylation sites related to inflammation and organ injury would be hypomethylated due to heat illness. Methods: Genomic DNA was extracted using the PAXgene Blood DNA Kit (Qiagen, Germantown MD, USA) from multiple timepoints from individuals who had heat exhaustion (n = 17), heat injury (n = 6), or EHS (n = 26). Genomic DNA (500 ng) was treated with sodium bisulfite using the Zymo EZ96 DNA Methylation Kit (Zymo Research, Orange CA, USA), and genome-wide DNA methylation patterns were profiled using the Infinium HumanMethylation450 BeadChip (850K) Kit (Illumina, Inc., San Diego CA, USA). All samples passed the log median intensity quality control of methylated and unmethylated channels assessed using the R minfi package v1.30.049. BMIQ normalization was applied on 99 EHS longitudinal samples. We utilized the ChAMP package for data normalization, filtering, and processing. The Short Time Series Expression Miner (STEM) was used to analyze patterns across three time points for EHS patients. STEM determines which profiles are statistically significant by comparing the actual number of genes assigned to a profile with the expected number of genes based on random chance. Profiles with more genes than expected are considered statistically significant, indicating that the expression pattern in those profiles is biologically meaningful. Ingenuity Pathway Analysis® was used to categorize pathways. Results: Epigenetic profiling of blood samples from individuals after collapse from heat illness revealed significant and distinct methylation changes, which were particularly pronounced in those suffering from EHS. In EHS samples, we identified 1,389 unique probes belonging to seven separate STEM profiles. Specifically, we found hypermethylation patterns related to detoxification of ROS, adipocyte signaling, insulin secretion signaling, and the WNT/B-Catenin pathway. We found hypomethylation patterns in the hepatic fibrosis pathway, serotonin pathway, and cytoskeleton signaling pathways, with the largest amount of hypomethylation appearing in Rho Family signaling. Genes such as RAB11FIP3, TTN, STAT5B, SAMSN1, PRG, LTBP1, PPARG, GPX3, ARHGEF3, and ITGAM, demonstrated hypermethylation, implicating disruptions in pathways responsible for maintaining structural integrity, metabolic homeostasis, and immune regulation. Additionally, hypermethylation of FAM109A and GNAZ, genes involved in neuronal signaling, suggests broader impacts affecting neural communication which is consistent with the altered mental status that is a hallmark of EHS at the time of collapse. Conversely, ZFP36L1, CDH11, and CH25H, exhibited hypomethylation, with roles in RNA stability, lipid metabolism, and extracellular matrix remodeling. Conclusion: Our results identify known and novel methylation patterns that may underpin the physiological and molecular responses to heat illness, particularly EHS. These insights provide a foundation for future research into the role of DNA methylation in mediating long-term health outcomes associated with heat injuries. Funded by USAMRDC; author views not representative of the Army or DoD. 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.