Exertional heat stroke causes long-term skeletal muscle epigenetic reprogramming, altered gene expression, and impaired satellite cell function in mice

Abstract

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.

Keywords: DNA methylation; RNA-Seq; calcium regulation; heat illness; satellite cell proliferation.

Figure 1.

A: maximum specific force in extensor digitorum longus (EDL) and soleus (SOL) muscles 1 mo after EHS exposure. n = 8 for EDL; n = 4 for SOL. Two-sample t test. B: [caffeine] required to induce contracture in both muscle groups. n = 8 in both groups. Data, nonparametric, central tendency expressed as median ±25–75% quartiles. Wilcoxon test. *P < 0.05. EHS, exertional heat stroke. EHS, exertional heat stroke; EXC, exercise control.

Figure 2.

Traditional volcano plot illustrating the number of mRNA species in EHS mice that reached a significant threshold of P < 0.05 and Log₂-fold change ≥ ±1 (FC > 2) compared with EXC (n = 8/group). Red: increased expression; blue: decreased expression. Significance determined by two-sample t test for parametric groups Wilcoxon for nonparametric groups. Listed genes are examples of affected genes. EHS, exertional heat stroke; EXC, exercise control.

Figure 3.

Two unbiased multivariate clustering and analysis estimates of the total dataset. A: hierarchical clustering [using Metaboanalyst (39)], allowing the algorithm to use the top 40 mRNA species that identify groupings. Data were normalized and auto-scaled before clustering, using Euclidean distance measures and the Ward clustering method [using Metaboanalyst (39)]. B: sparse PLS approach to obtaining principal components from all RNA species using sPLS-DA (using Metaboanalyst, graphed in SAS JMP 15.0 software, Cary, NC). Each vector for each animal was calculated using a maximum of 10 of the most significant variables. Both unbiased approaches demonstrate clear separation between EHS and EXC groups (n = 8/group). EHS, exertional heat stroke; EXC, exercise control.

Figure 4.

Gene ontology (GO) enrichment for biological processes of all significantly (P < 0.05) changed mRNA species in the dataset using Panther software (29). The Q value (FDR) is designated by color heat map. The fold enrichment is represented on the x-axis, and the size of the circles represents the number of genes represented in the specific biological process pathway.

Figure 5.

Heat maps of mRNA responses for individual EHS results, plotted as log2 of the fraction of the median of the EXC outcomes (n = 8/group). Boxes represent relative results for each assessed EHS animal. A: specific genes of interest were chosen from the entire set for their relationship to skeletal muscle contraction. B: specific genes of interest related to cell stress and to myogenesis during repair or remodeling. These specific pathways were chosen based on the results of GO Biological Process pathway analysis illustrated in Fig. 4. EHS, exertional heat stroke; EXC, exercise control.

Figure 6.

DNA methylation analyses of differentially methylated regions (DMRs). A: hypomethylation versus hypermethylation. B: genomic location of each DMR. C: KEGG pathway enrichment analysis. Significantly enriched KEGG pathways from DMR results using Network Analyst (28) are plotted with fold enrichment (x-axis). The color of the dots represents false discovery rate (FDR) and the size of the dots is based on the number of genes with DMRs assigned to the enriched KEGG pathway discovery rate. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 7.

Gene location of individual differentially methylated cytosines (DMCs) and differentially methylated regions (DMRs). A: scatterplot of individual DMCs [hypermethylation (red) or hypomethylation (black)] and DMRs [hypermethylation (green) or hypomethylation (blue)] as a function of distance from the transcription start site (TSS). B: histogram of the frequency of DMCs (black) and DMRs (red) as a function of distance from the TSS (±5,000 bp). Note: this is an expansion of the axis from A as indicated by the black dotted arrows. See text for details.

Figure 8.

Integrative Genomics Viewer (IGV) tracks of Tbx1 (A) and Pdha1 (B) after 30 days of recovery from EXC or EHS. The size of the region of the DNA and chromosome coordinates are indicated at the top of the figure. The two genome tracks indicate coverage for each sample (blue bars), i.e., the number of times that each CpG was sequenced (combined top and bottom strands). Only CpGs sequenced ≥10x are shown, with the scale set at 100 reads maximum. The middle track (intermittent thick blue areas) depicts the gene structure in which the thinnest, middle, and thickest lines indicate introns, UTR, and the coding exon, respectively. The direction of transcription is indicated by the arrowheads. The bottom two tracks indicate the percentage methylation (red bars) of all CpGs sequenced at each site ≥10x. Only sites represented by both samples were quantified for differential methylation. *Differentially methylated CpGs (DMCs) and differentially methylated regions (DMRs) demonstrating a statistically significant change in DNA methylation in EHS as compared with EXC are enclosed in cyan rectangles (solid and dashed lines indicate increased and decreased DNA methylation in EHS vs. EXC, respectively). DMCs were determined to be significant if they had a read depth of at least 10x per sample, a difference in methylation of ≥ ±10% with a false discovery rate (FDR)-corrected P value of Q < 0.05. A DMR was considered significant if it contained ≥ 5 CpGs within a maximum distance of 300 bp that all obtained changes in DNA methylation levels in the same direction (all CpGs with more or less methylation), a read depth of at least 10x per sample, an average change in methylation of ≥ ±10%, and a 2 D-KS (Kolmogorov–Smirnov test) P < 0.05. EHS, exertional heat stroke; EXC, exercise control.

Figure 9.

A: accumulated primary satellite cell counts of all EHS and EXC mice. Each box represents the number of cells in a given sample, binned into increasingly larger boxes (see legend for increments). The total binned value for each group is represented on the y-axis. n = 8 samples in each group, data analysis by two-way repeated-measures ANOVA showing significant group, time, and interaction effects. B: representative images for both EHS and EXC during proliferation assay at each timepoint. Note, the cell count images were modified together using the same brightness and contrast commands in Power Point software. EHS, exertional heat stroke; EXC, exercise control.

Figure 10.

A: fusion of primary satellite cells into myotubes (myogenic fusion index) was not found to be different between groups. Note, only five samples are shown in the EHS group because three EHS samples failed to proliferate and be incorporated into this assay. B: myotubes expressing myosin heavy chain in EHS and EXC groups. C: representative images for both EHS and EXC during myogenic assay. First column is stained nuclei with DAPI, second column is stained for myosin heavy chain imaged in Texas Red, and third column is the two columns merged. EHS, exertional heat stroke; EXC, exercise control.