Delayed metabolic disturbances in the myocardium after exertional heat stroke: contrasting effects of exertion and thermal load

Abstract

Epidemiological studies report higher risks of cardiovascular disease in humans exposed to heat stroke earlier in life. Previously, we explored mechanistic links between heat stroke and developing cardiac abnormalities using a preclinical mouse model of exertional heat stroke (EHS). Profound metabolic abnormalities developed in the ventricles of females but not males after 2 wk of recovery. Here we tested whether this lack of response in males could be attributed to the lower exercise performances or reduced thermal loads they experienced with the same running protocol. We systematically altered environmental temperature (T_e) during EHS to manipulate heat exposure and exercise performance in the males. Three groups of adult C57BL/6 male mice were studied: “EHS-34” (T_e = 34°C), “EHS-41” (T_e = 41°C), and “EHS-39.5” (T_e = 39.5°C). Mice ran until symptom limitation (unconsciousness), reaching max core temperature (T_c,max). After a 2-wk recovery, the mice were euthanized, and the ventricles were removed for untargeted metabolomics. Results were compared against age-matched nonexercise controls. The EHS-34 mice greatly elevated their exercise performance but reached lower T_c,max and lower thermal loads. The EHS-41 mice exhibited equivalent thermal loads, exercise times, and T_c,max compared with EHS-39.5. The ventricles from EHS-34 mice exhibited the greatest metabolic disturbances in the heart, characterized by shifts toward glucose metabolism, reductions in acylcarnitines, increased amino acid metabolites, elevations in antioxidants, altered TCA cycle flux, and increased xenobiotics. In conclusion, delayed metabolic disturbances following EHS in male myocardium appear to be greatly amplified by higher levels of exertion in the heat, even with lower thermal loads and max core temperatures.

NEW & NOTEWORTHY Epidemiological data demonstrate greater cardiovascular risk in patients with previous heat stroke exposure. Using a preclinical mouse model of exertional heat stroke, male mice were exposed to one of three environmental temperatures (T_e) during exercise. Paradoxically, after 2 wk, the mice in the lowest T_e, exhibiting the largest exercise response and lowest heat load, had the greatest ventricular metabolic disturbances. Metabolic outcomes resemble developing left ventricular hypertrophy or stress-induced heart disease.

INTRODUCTION

Several large retrospective population studies have followed the clinical histories of humans exposed to heat stroke over 12–14 years and have reported much higher incidences of developing cardiovascular disease and mortality compared with suitably matched controls (1–3). These correlative data could reflect a subgroup of individuals who, before heat stroke, have a propensity toward cardiovascular disease and were therefore more susceptible to heat stroke (4). Alternatively, the life-threatening stress of heat stroke could have been causative, leading to stress-induced, delayed-onset cardiovascular complications later in life.

Several years ago, in collaboration with US Army investigators, our laboratory developed a preclinical model of exertional heat stroke (EHS) in mice that simulates most of the clinical features seen in human EHS (5). Key outcomes include severe neurological symptoms and multiorgan injury (5, 6). This model allowed us to follow the pathophysiology over time during recovery. We discovered that the hearts of female mice, but not males, appeared to recover over the first week following EHS but then develop unexpected and profound myocardial metabolic disturbances and energy crises at 9–14 days (7). Male hearts exhibited near normal metabolomic profiles, with no histologic evidence of inflammation or injury. However, recent studies have reported similar EHS-induced myocardial injury in male rats following 2 wk of recovery (8); therefore, the phenomenon does not appear to be sex-specific in all rodents. In the current study, we set out to discover why the cardiovascular consequences were absent in male mice when compared with female mice. Could we reproduce the responses observed in females by altering the environmental conditions and exercise load during EHS?

One of the primary differences in the running responses of male and female mice in EHS is that female mice exhibit much greater exercise tolerances in hyperthermia, making it nearly impossible to replicate the exact nature of the environmental stress between females and males (9). For example, compared with males, under the same incremental exercise protocol, females greatly increase their exercise duration, speed, and time of exposure to high core temperatures. However, both sexes collapse at nearly identical peak core temperatures (T_c,max). We hypothesized that the greater integrated time of elevated T_c (i.e., thermal area or load) and/or the greater intensity of exercise in the heat, elevating strain on the heart, were responsible for the unique metabolic responses in females. To try to exclude one or more of these possibilities, we studied responses to EHS in males over a range of environmental temperatures (T_e) from 34°C, where the males could meet or exceed the exercise performance in females at 39.5°C, and at T_e = 41°C to attempt to accelerate the exposure and the intensity of the thermal load during the EHS exposure.

The results demonstrate that after a 2-wk recovery from EHS, males can exhibit very similar metabolic disorders in the heart compared with females. However, they require a more moderate hyperthermia exposure, allowing them to elevate their exercise intensity and duration sufficiently to induce a lasting effect on heart metabolism. Overall, the results provide evidence that the level of exertion during heat and not just heat load or absolute temperature is a critical variable in the pathogenesis of heat-induced metabolic disorders of the myocardium.

MATERIALS AND METHODS

Animal Subjects

This study was approved by the University of Florida’s Institutional Animal Care and Use Committee Protocol No. 201807422. Reported information about the procedures conformed to the “Animal Research Reporting of in vivo Experiments—ARRIVE guidelines” (10). All mice were male C57BL/6J (Jackson Laboratories, Bar Harbor, ME) and upon arrival, housed on a 12:12-h light/dark cycle at 22 ± 1°C and 30%–40% relative humidity. Standard chow (2919 Envigo; Teklad, Madison, WI) and water were provided, ad libitum. At the time of arrival, the mice were age-matched at 10-wk old and were ∼18 wk at the time of tissue collection. Mice were randomly separated into four groups of 8 mice, “EHS-34” (T_e = 34°C), “EHS-41” (T_e = 41°C), “EHS-39.5” [T_e = 39.5°C, our original EHS model (5)], and a group of naïve nonexercise controls (CNTR). Only males were studied in this cohort because the objectives of the study were to discover the reasons male and female mice were so metabolically different when exposed to the same incremental exercise stimulus and T_e, reported in our previous work (7, 9).

Emitter Implantation and Sham Surgery

Core temperature (T_c) telemetry devices (G-2 E-mitters, Star Life Sciences, Oakmont, PA) were implanted in the abdomen using sterile conditions, under isoflurane anesthesia (5.0% for induction; 0.5% for maintenance). Briefly, an incision was made on the shaved abdomen and an emitter was placed in the abdominal cavity as previously described (5, 6, 9). For postoperative pain, mice were treated with subcutaneous buprenorphine (0.1 mg/kg) injections every 12 h for 48 h. All mice were separated after surgery into single cages to eliminate the well-known effects of in-fighting among males. Naïve CNTR mice received identical sham laparotomy but with no emitter implanted. In CNTRs the abdominal cavity and peritoneal cavity were exposed for the same surgical duration as the other groups.

Exercise Training

Following a 2-wk recovery from surgery, mice were given in-cage running wheels (Model 0297-0521, Columbus Instruments, Columbus, OH, modified to fit the JAG75 ventilated cages, Allentown Caging, Allentown, PA) for voluntary wheel training. During the third week of training, all mice were brought to the laboratory and exercised on forced running wheels on 4 separate days (Lafayette, Model 80840, Lafayette, IN). The running wheels were modified to be powered by DC programmable power supplies, as described previously (5, 6). Briefly, on the first day, the mice were allowed to free-wheel on the forced running wheels, followed by a brief period of forced running at the slowest speeds. On the other 3 days the mice were immediately started on the forced running wheel program, beginning at the slowest speed of the EHS protocol, incrementing increases in speed every 10 min over an hour of exercise (see EHS Protocols). Following these four sessions, the mice were allowed to recover for ∼48 h, post training, before EHS. CNTR mice for this study did not go through any EHS or exercise training protocol. All EHS mice were brought to the laboratory on the evening preceding the EHS run. T_c was monitored continuously overnight and throughout the protocol by the implanted telemetry device at 0.5 min sample intervals. The mice were maintained overnight at laboratory room temperature ∼22°C in their own cages with food and water and kept on a 12 h timed light cycle, matching vivarium conditions. No mice had a core temperature >37.5°C during the hours before beginning the EHS protocol.

EHS Protocols

For all three protocols, each mouse was placed in its home cage within the environmental chamber (Thermo-Forma 3940 Incubator; Thermo-Fisher, Waltham, MA) before the run. The T_e was adjusted to 37.5°C for the EHS-37.5 group, identical to the original model that distinguished different responses between males and females (9). For the other models, the T_e was varied, based on pilot experiments. Two temperatures were selected that emphasized either the exertional component of EHS (EHS-34) or the thermal load component, (EHS-41). All EHS protocols began in the early morning at ∼0700 to 0730, as close as possible to the preceding active dark cycle (ends at 0700) and then the protocol proceeded as described previously (5, 6).

The incremental running protocols were the same for all three EHS groups. There were two phases: Phase 1, the “incremental phase,” mice began running at 3.1 m/min. Then the wheel speed was gradually increased by 0.3 m/min, every 10 min, until a core temperature (T_c) of 41°C was attained. Phase 2, the “steady state phase,” was reached when T_c exceeded 41°C, after which the running speed was maintained at a steady state until symptom limitation (unconsciousness). This also approximately corresponded to T_c,max. After reaching symptom limitation, as the mice recovered consciousness over ∼5–10 min, they were immediately placed back into their home cages and the environmental chamber returned to 22°C, which allowed all groups of mice to cool quickly. No mice died during or after any of these protocols, before euthanasia. The mice continued to be monitored visually for alertness and for return of T_c to normal values. After 4–6 h they were returned to the vivarium for a 2-wk recovery period. All voluntary running wheels were removed during this period. Thermal load was estimated from ongoing T_c measurements by integrating the area under the curve for the EHS trial, when T_c exceeded 39.5°C, i.e., $\int ({\text{T}}_{\text{c}}\times {\text{time),\hspace{0.17em}when\hspace{0.17em}T}}_{\text{c}}\text{\hspace{0.17em}> 39.5°C}$.

Tissue Collection and Metabolomics Analyses

On the 14th day, post EHS, mice were placed under isoflurane anesthesia and prepared for surgery. This was the end point for all animals in the study. Blood samples were drawn via transthoracic cardiac stick from under the xiphoid process, using a 27-gauge needle, preloaded with EDTA. Both ventricles of the myocardium were excised immediately, rinsed in ice cold buffer, and a transverse cut performed rapidly, separating the lower half from the upper half. The combined lower halves were immediately flash-frozen with liquid N₂, and stored at −80°C until shipment on dry ice for metabolomic processing and analysis by Metabolon Inc. (North Carolina).

The samples were prepared and analyzed at Metabolon Inc. using their standard protocols for the HD4 platform that used gas chromatography/mass spectrometry. Protein precipitation was done by placing samples in methanol and vigorously shaking, followed by centrifugation. The extract was then divided into five fractions: two were used for analysis by two separate reverse phase (RP) UPLC-MS/MS methods (ultrahigh performance liquid chromatography-tandem mass spectrometry), one with positive ion mode electrospray ionization (ESI) and one using negative ion mode ESI. Another fraction was used for hydrophilic UPLC-MS/MS with negative ion mode ESI; the remaining sample was reserved for backup. Samples were then extracted from their organic solvent and stored overnight before analysis. The next day, dried samples were reconstituted in solvents compatible with elucidating negative, positive, basic, and acidic compounds. All samples were run through a dedicated C18 column (Waters UPLC BEH C18-2.1 × 100 mm, 1.7 µm). Then mass spectrometry was performed and raw data files were produced. Raw data files were compared with Metabolon’s library of compounds and metabolites and their relative concentrations were determined as a fraction of the median of the CNTR samples. More than 800 different metabolites of various types were reported for each sample.

Statistical Analyses

Initial sample size per group was determined using G*Power. The “effect size” was determined using the difference in performance (distance run) in male mice from previous studies, resulting in a minimum “n” = 7.25. We therefore settled on an n = 8 for all groups. Raw outcomes involving performance during EHS were tested for normality using the Anderson-Darling test. All performance data proved to be parametric. The groups were compared using one-way ANOVA followed by post hoc analysis against the outcomes from the original EHS model (EHS-37.5) using the Dunnett’s method. No outliers or other data points were removed in any part of this study. All statistical analyses for physiological data were performed using SAS-JMP Pro 15-17.

Metabolites determined from the HD4 platform were initially quantified, normalized, and interpolated by Metabolon. The investigators then renormalized the sample sets from all three EHS groups to a fraction of the median from the CNTR mice. Therefore, all groups were expressed as a fraction of CNTR. Data was then analyzed using the online metabolomics analysis tool, MetaboAnalyst 5.0 (RRID:SCR_015539) (11) and SAS JMP 17. The individual methodologies and details used with MetaboAnalyst 5.0 are described in accompanying figure legends. P < 0.05 was considered statistically significant, but each comparison was evaluated for its potential false discovery rate using Benjamini and Hochberg (12) and the resulting Q values for significance scaled for the list of significant findings in each experimental group. No outliers or other metabolic data points were removed, and no transformation procedures were performed on the metabolomics outcomes.

RESULTS

Performance

Individual core temperature profiles, over time, up to symptom limitation, for each of the three EHS models are illustrated in Fig. 1. At the lower environmental temperature, the EHS-34 mice ran for much longer durations compared with the other two models. During the midpart of the EHS exposure, the EHS-34 group was able to compensate for the increasing heat production as exercise intensity increased, indicated by a steady or decreasing T_c between ∼60 and 180 min of the run (blue lines, Fig. 1A). In contrast, the EHS-37.5 and the EHS-41 groups had very similar T_c profiles that exhibited only brief plateau periods before an exponential rise in T_c until symptom limitation was attained.

Figure 1.

Minute-by-minute core temperature profiles in all groups of animals exposed to the exertional heat stroke protocol; EHS-34 (EHS group exercised in 34°C), EHS-41 (EHS group exercised in 41°C), and EHS-37.5 (original model of EHS, mice exercised in 37.5°C). A: profiles of EHS-34 against EHS 37.5°C. B: profiles of EHS-41°C exposures compared with 37.5°C exposures. EHS, exertional heat stroke.

Grouped data for key physiological responses to the different EHS heat exposures are shown in Fig. 2. On average, the EHS-34 mice ran in the heat ∼2 times longer than the EHS-37.5 mice (averaging ∼5 h) (Fig. 2A); they ran nearly three times the total distance (Fig. 2B) and they achieved ∼2 times the peak running speed (Fig. 2C). These responses were higher than the original observations in females [light green box plots in Fig. 2, redrawn from historical data from our laboratory (9)]. The mice reached symptom limitation (unconsciousness) at a significantly lower T_c,max in the EHS-34 group (Fig. 2D) and experienced only ∼1/3 of the total thermal load, as measured by the thermal area (Fig. 2E). Interestingly, the EHS-41 group did not exhibit significantly different exercise responses compared with the EHS-37.5 group, reached symptom limitation at a very similar T_c,max (Fig. 2D), and were exposed to similar thermal loads over the course of the trial compared with EHS-37.5 mice (Fig. 2E).

Figure 2.

Physiological responses to EHS using the three experimental protocols. A: running time to T_c,max (the maximum core temperature acquired at symptom limitation). B: the total distance run during the EHS protocol. C: the maximum running speed attained during the EHS protocol. D: the maximum core temperature achieved during the EHS protocol. E: the total thermal area acquired during the EHS protocol (i.e., the integration of time and core temperature >39.5°C). EHS-37.5 = the original EHS protocol (7), EHS-34 (T_e = 34°C), EHS-41 (T_e = 41°C). n = 8 mice/group, means ± SD. All data expressed as fraction of control mice (CNTR). Statistics: one-way ANOVA followed by Dunnett’s postdoc against the EHS-37.5 data. *P < 0.05, ***P < 0.001, ****P < 0.0001. Green box plots represent mean and SD from previous data published using EHS-37.5 (9). EHS, exertional heat stroke.

Metabolic Responses of the Ventricular Myocardium

Comparisons of the specific metabolites that were both significantly different and that showed >1.5-fold deviation from the CNTR group are displayed in traditional volcano plots in Fig. 3. The EHS-37.5 group (Fig. 3) exhibited only modest changes in metabolites, most of which were decreased in abundance (blue-green symbols). This aligns with our previous metabolomic analyses of male hearts using this T_e (7). In contrast, both the EHS-34 (Fig. 3A) and EHS 41 (Fig. 3B) groups showed abundant increases in important metabolites with a few decreases reaching significance (Fig. 3, B and C). Interestingly, nearly all metabolites that met the cutoff significance criteria in the EHS-41 group (Fig. 3C), were also represented in the EHS-34 responses (Fig. 3A). However, many more metabolites in the EHS-34 group were increased over CNTR and, in general, reached higher levels of significance and fold change.

Figure 3.

Volcano plots for each metabolite in the EHS, exertional heat stroke (EHS) groups tested against a common control (CNTR), 2 wk after EHS. A: changes in myocardial metabolites when environmental temperature T_e was 34°C (EHS-34). B: changes in myocardial metabolites when T_e = 37.5°C (EHS-37.5). C: changes in myocardial metabolites when T_e = 41°C (EHS-41). Metabolite species that were statistically significant (P < 0.05) and exhibited greater than ±1.5 fold change in concentration relative to CNTR animals are highlighted in blue/green (decreased) and red (increased). Data were analyzed using the MetaboAnalyst 5.0 tool and recreated using GraphPad Prism 9.0. EHS-37.5 = the original EHS protocol (7), EHS-34 (T_e = 34°C), and EHS-41 (T_e= 41°C). CNTR, control.

We further compared these global untargeted metabolomic responses in a variety of classical ways. These included a variant of principal component analysis (PCA) called the “sparse partial least squares discriminant analysis” (sPLCA; 13), which very effectively reduces noise from variables that do not discriminate well between groups. It is an improved approach to evaluating groups with large numbers of variables. Results for sPCLA plots are shown for three primary components in Fig. 4, with net vector coordinates represented as symbols for each animal sample, colored by group. Although all groups separated from the other groups, the EHS-34 group clearly deviated the most from all other groups, particularly along the component 1 dimension. The geometric separation between CNTR and the EHS-37.5 groups was the smallest. Chief loading metabolites for each of the 3 components associated with each experimental group are provided in Supplemental Fig. S1.

Figure 4.

Unbiased principal components, calculated from a maximum of 40 metabolites in each group to establish each component using the “sparse partial least squares discriminant analysis” approach (sPLSDA; 13). All groups clearly separated, but the strongest separation was seen in the EHS-34 group (blue dots). Each point represents the resultant three-coordinate vectors from one heart sample. Coordinate data was calculated by MetaboAnalyst 5.0, and raw vector data was used by SAS JMP 16.0 for the final 3-D graphical display. CNTR, control; EHS, exertional heat stroke; 3-D, three-dimensional.

The data were also analyzed by traditional unbiased hierarchical analyses and heat maps, with each model compared with the CNTR (Supplemental Fig. S2). Unbiased hierarchical analyses could also clearly identify EHS-34 and EHS-41 when compared with hearts from CNTR. However, the analyses could not clearly identify samples from the EHS-37.5 group from the CNTR samples (data not shown).

All statistically significant metabolites (P < 0.05), irrespective of fold change, were also compared with CNTR samples by rank order of the P value in forest plots for EHS-34 (Supplemental Fig. S3), EHS-41 (Supplemental Fig. S4), and EHS-37.5 (Supplemental Fig. S5). In each of these relationships, the evaluation of the possible error due to multiple comparisons is expressed on the right y-axis, using the Benjamini–Hochberg Q value. Note that 26 metabolites reached a Q threshold of < 0.2 in the EHS-34 group but only one variable in either the EHS-41 and EHS-37.5 reached Q < 0.2. This emphasizes the greater effect size of the metabolic changes seen in the hearts of EHS-34 mice compared with the other two groups.

To assign physiological meaning to these observations, we selected sets of metabolites that provide some insight into potential underlying pathophysiology, as it relates to known cardiovascular disorders. In Fig. 5A, metabolites involved in carbohydrate metabolism were clearly elevated in the EHS-34 group, but not in the other groups. For metabolites reflecting mitochondrial and TCA cycle function (Fig. 5B), both isocitrate and succinate were elevated in EHS-34, representing critical points in TCA cycle flux. Curiously, creatine phosphate was significantly elevated, which suggests that the mitochondria were functioning sufficiently to support energy production. This is an opposite result from what we previously observed in females at this time point (7). AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) was also elevated, which is an endogenous form of the pharmacological drug used to chemically stimulate AMPK (14). Fat metabolism in all three EHS models showed a generalized reduction in acylcarnitines and some increases in other forms of lipid storage and lipid metabolites, which roughly aligns with our previous observations in females (7).

Figure 5.

Scatterplot of fold changes in specific metabolites related to carbohydrate metabolism (A), mitochondrial metabolism (B), and fatty acid metabolism and lipids (C). Statistics are t tests or Wilcoxon signed ranks for parametric or nonparametric data, respectively, against CNTR. The vertical solid lines are medians; the dotted line represents the median of the CNTR values for that metabolite. n = 8 in each group. CNTR, control.

Figure 6 illustrates the representative changes that were observed in amino acid metabolism, redox balance, and ongoing damage and repair. In Fig. 6A, EHS-34 and EHS-41 exhibited elevations in a number of amino acid metabolites, particularly related to branched-chain amino acids, characteristic of some forms of cardiac disease (15, 16). Figure 6B illustrates the metabolites in EHS-34 that are consistent with the myocardium being poised in a state of antioxidant defense, with a large elevation in glutathione, cysteine, ascorbate, and their metabolites. In Fig. 6C, the metabolite, N1-methyl-2-pyridone 5-carboxamide (2PY), considered a xenobiotic is elevated in the EHS-34 and EHS-41 groups. It has been identified as a uremic toxin (17) and is a byproduct of NAD(H) degradation. Heme reductions were observed in the EHS-34 hearts that may reflect the ongoing activity of heme oxygenase as another potent antioxidant defense mechanism. The metabolite, pro-hydroxy pro (prolyl hydroxyproline) was also elevated in EHS-34. It is a by-product of collagen degradation and is commonly associated with tissue remodeling (16). Trimethylamine N-oxide (TMAO) was increased in both EHS-34 and EHS-41 groups, a xenobiotic originating in the gut and known to be elevated in several cardiovascular diseases (18).

Figure 6.

Scatterplot of fold changes in specific metabolites related to amino acid metabolism (A), redox balance (B), and stress, injury, and repair (C). Statistics are t tests or Wilcoxon signed ranks for parametric or nonparametric data, respectively, compared with CNTR. The vertical solid lines are medians; the dotted line represents the median of the CNTR for that metabolite. n = 8 in each sample. CNTR, control.

In Table 1, significantly altered metabolites of interest in the EHS-34 group, with a ±1.5-fold change, are listed on the left column. Results are compared with male EHS-41 mice (second column) as well as from the historical female study using the EHS-37.5 model (7) in the third column. Notably, 73% (35/48) of the significantly altered metabolites that were observed in the male EHS-34 group were significantly changed in at least one of the other two experimental groups, mostly in the male EHS-41 and in female EHS-37.5 mice (9). These results demonstrate that even across wide-ranging environmental exposures, sex and exercise conditions, there are many common metabolic disturbances in the ventricles that emerge at this time point following EHS exposure.

Table 1.

Common metabolites observed in male and female models of EHS

Functional Class	Male EHS-34	Male EHS-41	Female EHS-34
Shift to glucose metabolism	↑glucose, ↑pyruvate, ↑maltose, ↑maltotriose, ↑fructose, ↑mannitol/sorbitol	↑glucose	↑maltose
Disordered glycolysis, pentose pathway, glycerophosphate shuttle	↑glycerol 3-phosphate		↓glycerol 3-phosphate
Disordered TCA cycle metabolites	↑succinate, ↑isocitrate		↓succinate, ↑isocitrate
Shift from lipid metabolism	↓acylcarnitines, ↑acylglycines, other FA derivates, ↑pantetheine	↓acylcarnitines, ↑acyl glycines, ↑other FA derivates	↓acylcarnitines, ↑pantetheine, ↑other FA derivates
Increased amino acid metabolism	↑pro-hydroxy-pro, 2-hydroxy-3-methylvalerate, ↑S-methyl methionine, ↑threonate, ↑N-acetylleucine, ↑cysteine, ↑alpha-hydroxyisocaproate, ↑4-guanidinobutanoate	↑pro-hydroxy-pro, ↑S-methyl methionine, ↓isoleucyl-hydroxyproline	↑S-methyl methionine, ↓N-acetylleucine, ↑cysteine
Response to oxidative stress	↑glutathione, ↑cysteine, ↑ascorbate, ↑ascorbic acid 3-sulfate, ↑dehydroascorbate, ↓heme, ↑spermine, ↑lactoylglutathione		↑glutathione, ↑dehydroascorbate, ↑cysteine, ↓heme, ↓spermine, ↑lactoylglutathione
Xenobiotic or plant derived mediators of cardiovascular disease	↑trimethylamine N-oxide, ↑imidazole propionate, ↑thioproline, ↑3-(3-hydroxyphenyl) propionate sulfate, ↑3-(4-hydroxyphenyl)propionate, ↑N-acetyl-2-aminooctanoate, ↑dihydroferulic acid sulfate	↑imidazole propionate, ↑trimethylamine N-oxide, ↑N-acetyl-2-aminooctanoate	↑trimethylamine N-oxide, ↑3-(3-hydroxyphenyl) propionate sulfate
Other unique markers	↑AICAR, ↑N1-methyl-4-pyridone-3-carboxamide, ↑creatine phosphate, ↑pantetheine, ↑phospho-pantetheine, ↑5-methyltetrahydrofolate, ↑corticosterone,↑(3′-5′)-adenyladenosine, ↑1-stearoyl-2-docosahexaenoyl-glycosyl-GPE	↑AICAR, ↑3′-5′adenylyladenosine, ↑nicotinamide-N oxide	↑N1-Methyl-4-pyridone-3-carboxamide, ↓creatine phosphate, ↑nicotinamide-N oxide, ↑1-stearoyl-2-docosahexaenoyl-glycosyl-GPE, ↑pantetheine and ↑phospho-pantetheine

Significant metabolites in EHS-34, EHS-41, and female EHS-37.5 [from previous study (7) that are functionally related to heart disease]. Key: the table compares the significant metabolites in the EHS-37.5 with similar significant metabolites in the EHS-41 group and the females EHS-37.5 historical group from (7). Note, some other metabolites in the female EHS-37.5 study were significant but not represented in the male groups because of a different metabolomics library. They are not shown here. EHS, exertional heat stroke; FA, fatty acid. Bold metabolites: significantly altered in two of the three groups. Underlined metabolites: increased metabolites. Italicized metabolites: significantly decreased in that group but also represented in another column or group. Up arrows (↑) signify increased levels of metabolite; down arrows (↓) signify decreased levels.

DISCUSSION

The results provide evidence that metabolic disturbances in the myocardiums of male mice are extensive but require sufficiently high levels of exertion and/or thermal load for them to become evident. Interestingly, it was the EHS-34 group that displayed the most severe metabolic responses to EHS, a group exposed to the lowest T_e, the lowest thermal load, and the lowest T_c,max. This finding suggests that the magnitude or duration of heat exposure is not the only variable driving myocardial pathology in this setting. The results support a critical role of the overall “strain” placed on the heart in the presence of hyperthermia and exercise that causes the disorder(s).

As summarized in Fig. 7, the metabolomics results, particularly in the EHS-34 group, are characterized by: 1) an apparent shift to carbohydrate metabolism and reduced lipid metabolism, 2) a substantial increase in substrates used in antioxidant defense, suggesting a recent history of oxidant stress, 3) accumulation of metabolites from the gut or kidney that have been taken up by the heart and that could contribute to heart pathology (17, 19), 4) an increase in amino acid metabolism, and 5) apparent bottlenecks or alterations in TCA cycle carbon flux. These outcomes suggest the emergence of a unique type of post heat stress-induced metabolic heart dysfunction that resembles the early development of ventricular hypertrophy (16, 20), or a transition state to other forms of stress-induced pathology (21). This metabolic outcome is often referred to as a loss of “metabolic flexibility” in the cardiovascular literature (22) and may reflect other ongoing whole body metabolic changes leading to alterations in body mass, appetite, and development of metabolic syndrome. This ultimately may also be linked to epigenetic responses to severe heat exposure (23), recently described by our laboratory in female hearts, 1 mo following EHS (24).

Figure 7.

Summary of categories of metabolic disorders based on metabolite alterations in the ventricles of the heart following exertional heat stroke. Created with BioRender.

Combined Effects of Exertion and Environmental Heat during EHS

The physiological results emphasize the importance of the exertional component of EHS in both the onset of severe neurological symptoms and the progression to organ damage. Unique aspects of the stress induced by exertion during EHS includes the redistribution of cardiac output to support exercising muscles and the myocardium on top of the cardiac output needed for elimination of heat, thus further reducing venous return, reducing tissue nutrient flow to vital organs (25, 26). This constitutes an elevation in “cardiac strain.” Though in clinical settings, the strain on the heart most often refers to mechanical deformation (27), we infer its use here in a broader context that combines the mechanical strain and the metabolic strain resulting from nutrient depletion and rapidly increasing energetic costs of contraction. In humans, markedly increased cardiac strain during exertional hyperthermia has been clearly described with accompanying declines in stroke volume, arterial pressure, and central venous volume (28). Passive heat stress in humans shares some of these characteristics, particularly the loss of central venous volume and reduced end-diastolic volume (29, 30). It is possible that such strain on the heart may lead to structural and inflammatory changes in the myocardium that have been observed in humans using magnetic resonance imaging, as long as 3 mo after exposure to heat illness (31).

In most preclinical models of nonlethal heat stroke in mice (exertional and classic), symptom limitation or organ injury occurs between T_c,max, of ∼42.2°C and 42.5°C (5, 9, 32), which is consistent with the idea that the neurological crisis at symptom limitation and/or organ injury are due to a common “high temperature threshold” phenomenon. However, the greatest effects on the heart in this study were observed in the EHS-34 mice, which experienced the lowest environmental temperature, the lowest T_c,max (∼41.8°C), and the lowest thermal load. This suggests that other variables such as our broad definition of cardiac stain during hyperthermia may be as important to the development of heart pathology and possibly the neurological symptoms at maximum T_c. The animal group that experienced the highest overall thermal load (EHS-41) must have also experienced a considerable strain on the heart, as these mice had to elevate cardiac output even further to compensate for the higher T_e. This may help to understand why this group also showed considerable disturbances in heart metabolomics. In contrast, in the male EHS-37.5 model in both this and our previous study (7), there was little evidence of sustained metabolic disorders, suggesting that the physiological demands to meet the needs of both temperature regulation and exercise in this group were insufficient to induce lasting disturbances. It is likely that the physiological response induced in females at EHS-37.5 was due to their remarkable capacity to exercise with greater intensity and duration in a given temperature compared with males (9), putting a greater relative strain on the heart.

An additional aspect of more extreme exercise in the heat that might contribute to the results in the EHS-34 mice is that these animals were likely pushing the limits of nutrient delivery, particularly when the exercise period is prolonged and glycogen stores are depleted. This puts strain on the metabolic flux of energy in the heart and other tissues. Furthermore, byproducts of muscle contraction and muscle metabolism, e.g., lactate, H⁺, K⁺, ADP, Pi, and NH₃, in combination with hyperthermia, could compromise metabolic and contractile function in the heart or other organ systems.

Highlights and Significance of Ventricular Metabolomic Responses to EHS

A summary of the overall significant metabolomic changes in the ventricles of male EHS-34, male EHS-41, and female EHS-34 mice [from our previous study (7)], are summarized in Table 1. The male EHS-37.5 mice had very few metabolites that changed significantly and were therefore not included in Table 1. A predominant finding within the EHS-34 males was an increase in metabolites related to glucose as a metabolic substrate and a shift away from fatty acid metabolism (Table 1). Elevations of tissue glucose and pyruvate, as well as maltose and maltotriose, (intermediate metabolites in glycogenolysis) are typical of results from studies of heart hypertrophy (33, 34). The increase in sorbitol and fructose are indicators of activation of the “polyol” or aldose reductase pathways, also activated in hypertrophied, ischemic, and failing hearts, which can serve secondarily to stimulate lipid accumulation (20). This was also clearly observed in female hearts following EHS-37.5 (7). In both male EHS-34 and female EHS-37.5 groups, there were changes in the movement of glucose substrates to alternative glycolysis pathways. In males, based on elevations in glycerol 3-phosphate, there appears to be a shift toward the glycerol-phosphate shuttle that replenishes NAD+ for mitochondrial metabolism (35). In contrast, in females, there was a depression of glycerol-3 phosphate and an elevation in other metabolites involved in the pentose phosphate pathway. This pathway rescues NADPH for recovery of antioxidant enzyme systems and other redox-regulating enzymes (36). The energetic states of the heart in males and females also differed. For example, in male EHS-34 mice, creatine phosphate was elevated (Fig. 5B) and NAD(H) was normal (not shown), whereas in females, using the EHS-37.5 model (7), creatine phosphate and NAD(H) were greatly depleted, suggesting an extreme energy crisis. One possible mechanism for these sex differences was the elevated AICA ribonucleotide, seen at this timepoint only in the male EHS-34 and EHS-41 hearts. This activator of AMPK could function as a rescue pathway for energy metabolism to sustain energy stores until normal pathways of flux can be restored (37).

Elevations in isocitrate were observed in both the male EHS-34 and in the female EHS-37.5 model. Increased isocitrate can indicate a slowdown of flux through isocitrate dehydrogenase within the TCA cycle, an enzyme highly sensitive to oxidative stress and damage, and an early metabolic precedent in the development of cardiac hypertrophy (38). Succinate was increased in the male EHS-34 mice but reduced in the female mice, suggesting some sex-dependent differences on the TCA cycle (Table 1). Interestingly, elevations in mitochondrial succinate can result in elevated mitochondrial reactive oxygen formation and therefore could underlie a mechanism for the strongly expressed antioxidant responses in male EHS-34 hearts (39).

A consistent response across all models of male EHS and in our previous experience with the female EHS were marked reductions in acylcarnitines of all forms. In this male EHS study, we did not have the wide spectrum of different acylcarnitines or other lipid species to evaluate because we did not use the lipidomics platforms that were used in the original female study (7). Considering the few available acylcarnitines that were measured in this library, there were marked decreases across all models. Acylcarnitines are created from free fatty acids to pass lipid substrates for energy production into the mitochondrial matrix via carnitine palmitoyl transferase 2 (CPT2) on the inner mitochondrial membrane. The marked reduction in acylcarnitines is consistent with left ventricular hypertrophy, in experiments using a transverse aortic constriction (16) and in other models of advanced heart failure (40). We speculate that the response of acylcarnitines reflects a decrease in substrates needed for β-oxidation, though it could also mean that acylcarnitines are being rapidly metabolized as fuel faster than they can be replenished.

The effects of these EHS models in male mice on other lipid species are less conclusive because we had only a limited number of metabolic species identified in the library. By comparison, in our previous study in females, where lipidomics was performed, lipid species of many forms were greatly elevated (7). We suspect lipids were also accumulating in the male hearts in EHS-34 hearts based on a few species of acylglycines available, which were increased in concentration, see Fig. 5C. We also observed marked increases in pantetheine and phospho-pantetheine in both EHS-34 and female EHS-37.5 hearts, which are essential cofactors in fatty acid synthesis (41).

Amino acid metabolites, particularly metabolites of branched-chain amino acids, leucine, isoleucine, and valine were elevated; these are also characteristic of metabolomic profiles of left cardiac hypertrophy and heart failure (34, 42). There was extensive evidence for this in our male EHS-34 group (Fig. 5). Other amino acid derivatives involved in methionine metabolism such as methionine 5-methyltetrahydrofolate (5MeTHF), and S-methylmethionine were elevated, and these or related metabolites have been observed in some forms of heart hypertrophy (16). Other metabolites, peptides, or amino acid derivatives involved with oxidative defense were increased in EHS-34 group, including high levels of reduced glutathione (an antioxidant response) and its closely aligned redox partner, cysteine (43), s-lactoylglutathione and products of ascorbate metabolism (7). These outcomes suggest an ongoing activation of antioxidant defenses. An interesting dipeptide, prolyl hydroxyproline (pro-hydroxy-pro), was also elevated and reflects ongoing collagen degradation and is used as a marker of myocardial remodeling in overloaded hearts (16). Another metabolite that may be indirectly related to defense of oxidative stress, is the reduction in heme in both the male EHS-34 and EHS-41 mouse models (Fig. 6C) and in the females using the EHS-34 model from our previous study (7). Heme, within the myocytes, exists as part of the electron transport chromophores, as a component of myoglobin and in small amounts as free heme. Free heme can be toxic, and metabolism of heme by heme oxygenase is of recognized importance as an antioxidant system that scavenges oxidants and produces carbon monoxide, also providing protective functions in inflammatory environments (44).

Besides triethylamine and N1-methyl-2-pyridone-5-carboxamide described earlier, other interesting xenobiotics were identified such as thioproline and dihydroferulic acid sulfate, both of which have significant antioxidant and/or anti-inflammatory properties (45, 46). The exact links between intestinally derived or uremia-derived metabolites and heart function are poorly understood, but considering that both the intestinal barrier and the kidney are prime target organs for the effects of heat stroke in EHS (5), the observation may provide a possible direction for future research. Finally, the remarkably elevated corticosterone seen only in the EHS-34 males (Fig. 6C) may be another key to the unravelling of the altered metabolic substrate control; this change was not seen in the females or any other male EHS model we have studied. Epigenetic responses to heat that involve corticosterone signaling are an active area of research, particularly with respect to metabolic resilience and tolerance to subsequent heat exposures (47).

Study Limitations

There are a number of important limitations to the approach taken by this study. First of all, the human and rodent thermoregulatory systems are quite different and rely on contrasting compensatory systems. For example, mice do not substantially sweat, though they can lose up to 5%–10% of body water during EHS (5). Much of the heat exchange occurs through the ears, the tail, and through evaporative heat loss by grooming with saliva (48, 49). Furthermore, their thermoneutral zone is much higher, being centered ∼30°C–32°C, whereas our mice were acclimatized to 22°C in their home cages. Maintaining a normal core temperature in mice at 22°C requires a several-fold elevation in metabolic rate (48), and it is likely they would respond to heat differently had they been acclimatized within their thermoneutral zone. In addition, mice are normally active in the dark cycle whereas we studied these mice in their early light cycle, when they normally sleep. Nevertheless, all groups were exposed to the same environmental conditions in terms of the EHS intervention and the resting temperature, so we do not believe this is a major limitation for comparisons between groups.

Stress, superimposed on both heat and exercise, could contribute to the development of heart abnormalities because stress hormones, catecholamines (50), and corticosteroids (51), have strong influences on the development of a variety of cardiovascular diseases. From our perspective, some aspects of the acute stress inherent to this protocol in mice may parallel stress conditions of humans during life-threatening heat exposures. For example, in both man and mouse, corticosteroid levels are acutely increased in heat stress (52, 53). Therefore, stress response hormones or other aspects of stress exposure during hyperthermia may play a role in the metabolic effects on the heart and other organ systems.

Conclusions and Translational Implications to Heat Medicine

Coupled with previous work in female mice (7), we have provided evidence that a single exposure to EHS, now shown in both male and female mice, can induce sustained cardiovascular metabolic disorders that may impair exercise capacity, lead to apparent loss in heat tolerance during recovery and possibly lead to longer term cardiovascular dysfunction and disease. This is an emerging phenomenon that we are just now beginning to study. Clearly, there remain an enormous number of questions to ask by future studies and future investigators, including unraveling the link between cardiac strain, injury, and metabolism in the heat, questions regarding the ubiquitousness of the metabolic findings to other tissues, the effects on long-term mitochondrial function, the source of oxidative stress and the functional significance of the epigenetic changes we have observed in different tissues in response to these heat stress exposures (24). Though the metabolic phenotype and pathology appear to resemble the development of early left ventricular hypertrophy, it is noteworthy that it is still heterogenous and depends on the level of exercise and heat exposure experienced by the mice. At this 2-wk recovery interval, what we are likely observing is a transition state in which the heart is undergoing metabolic remodeling to return to some form of steady-state metabolism. This calls for more long-term recovery studies to reveal the chronological progression or eventual repair from this disorder. Based on emerging literature in heat-stroke-related heart disease, it is at least possible that this phenomenon in mice is clinically relevant to the human condition, but it will take further work to fully understand how it manifests itself in limiting cardiovascular capacity, altering endothelial function, hypertension, heart mechanics, etc. It is possible that by understanding the underlying nature of this disorder in animals, we will be able to apply the data to humans to develop preventative and treatment paradigms that will avoid such long-term cardiovascular consequences.

DATA AVAILABILITY

Raw data used to generate figures and analysis are available at https://original-ufdc.uflib.ufl.edu//IR00012122/00001.

SUPPLEMENTAL DATA

Supplemental Figs. S1–S5: https://doi.org/10.6084/m9.figshare.23499105.v3.

GRANTS

US Army Medical Research and Development Command BA180078, W81XWH015-2-0038 (to T.L.C.), and the University of Florida Foundation BK Betty Stevens Professorship (UF Foundation) Grant F00294 (to T.L.C.). J.M.A. was supported by a training fellowship from the Department of Exercise Physiology, College of Sport Sciences and Physical Activity, King Saud University, Kingdom of Saudi Arabia. G.P.R. was supported by NIH 5T32HL134621 BREATHE training fellowship directed by Gordon S. Mitchell at the University of Florida.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.K.G. and T.L.C. conceived and designed research; C.K.G., B.J.G., G.P.R., M.T.R., and J.M.A. performed experiments; C.K.G. and T.L.C. analyzed data; C.K.G. and T.L.C. interpreted results of experiments; C.K.G. and T.L.C. prepared figures; C.K.G. and T.L.C. drafted manuscript; C.K.G., B.J.G., G.P.R., M.T.R., J.M.A., and T.L.C. edited and revised manuscript; C.K.G., B.J.G., G.P.R., M.T.R., J.M.A., and T.L.C. approved final version of manuscript.