The intensity-dependent effects of exercise and superimposing environmental heat stress on autophagy in peripheral blood mononuclear cells from older men.

Autophagy is a vital cellular process, essential to maintaining cellular function during acute physiological stressors including exercise and heat stress. We previously showed that autophagy occurs during exercise in an intensity-dependent manner in peripheral blood mononuclear cells (PBMCs) from young men, with elevated responses in the heat. However, given autophagy declines with age, it is unclear whether a similar pattern of response occurs in older adults. Therefore, we evaluated autophagy and the cellular stress response [i.e., apoptosis, inflammation, and the heat shock response (HSR)] in PBMCs from 10 healthy older men [mean (SD): aged 70 yr (5)] in response to 30 min of semirecumbent cycling at low, moderate, and vigorous intensities [40, 55, and 70% maximal oxygen consumption (V̇o2max), respectively] in a temperate (25°C) environment, with an additional vigorous-intensity bout (70% of V̇o2max) performed in a hot environment (40°C). Responses were evaluated before and after exercise, as well as throughout a 6-h seated recovery period performed in the same environmental conditions as the respective exercise bout. Proteins were assessed via Western blot. Although we observed elevations in mean body temperature with each increase in exercise intensity, autophagy was only stimulated during vigorous-intensity exercise, where we observed elevations in LC3-II (P < 0.05). However, when the same exercise was performed in the heat, the LC3-II response was attenuated, which was accompanied by significant p62 accumulation (P < 0.05). Altogether, our findings demonstrate that older adults exhibit autophagic impairments when the same vigorous-intensity exercise is performed in hot environments, potentially underlying heat-induced cellular vulnerability in older men.NEW & NOTEWORTHY We demonstrate that autophagic stimulation occurs in response to short-duration (30-min) vigorous-intensity exercise in peripheral blood mononuclear cells from older adults; however, no changes in autophagy occur during low- or moderate-intensity exercise. Moreover, older adults exhibit autophagic impairments when the same vigorous-intensity exercise is performed in hot ambient conditions. When paired with an attenuated heat shock response, as well as elevated apoptotic responses, older men may exhibit greater cellular vulnerability to exertional heat stress.

Changes in surrogate markers of intestinal epithelial injury and microbial translocation in young and older men during prolonged occupational heat stress in temperate and hot conditions.

PURPOSE: Exertional heat stress can cause damage to the intestinal epithelium and disrupt gastrointestinal barrier integrity, leading to microbial translocation (MT) linked to the development of heat stroke. This study aimed to assess age-related differences in markers of intestinal epithelial injury and MT following non-heat stress and high-heat stress exercise in healthy young and older men. METHODS: Markers of intestinal epithelial injury (intestinal fatty acid-binding protein-'IFABP') and MT (soluble cluster of differentiation 14-'sCD14'; and lipopolysaccharide-binding protein-'LBP') were assessed in healthy young (18-30 y, n = 13) and older (50-70 y) men (n = 12). Blood samples were collected before, after 180 min of moderate-intensity (metabolic rate: 200 W/m2) walking and following 60 min recovery in either a non-heat stress [temperate: 21.9 °C, 35% relative humidity (RH)] or high-heat stress (hot: 41.4 °C, 35% RH) environment. RESULTS: There were no differences in IFABP and sCD14 between the young and older groups in the temperate condition, while LBP was greater in the older group (+ 0.66 ug/mL; + 0.08 to + 1.24 ug/mL). In the hot condition, the older group experienced greater increases in IFABP compared to the young group (+ 712 pg/mL/hr; + 269 to + 1154 pg/mL/hr). However, there were no clear between-group differences for sCD14 (+ 0.24 ug/mL/hr, - 0.22 to + 0.70 ug/mL/hr) or LBP (+ 0.86 ug/mL/hr, - 0.73 to + 2.46 ug/mL/hr). CONCLUSION: While older men may experience greater intestinal epithelial injury following exercise in the heat; this did not lead to a greater magnitude of microbial translocation relative to their younger counterparts.

Impaired autophagy following ex vivo cooling of simulated hypothermic temperatures in peripheral blood mononuclear cells from young and older adults.

Hypothermia is a critical consequence of extreme cold exposure that increases the risk of cold-related injury and death in humans. While the initiation of cytoprotective mechanisms including the process of autophagy and the heat shock response (HSR) is crucial to cellular survival during periods of stress, age-related decrements in these systems may underlie cold-induced cellular vulnerability in older adults. Moreover, whether potential sex-related differences in autophagic regulation influence the human cold stress response remain unknown. We evaluated the effect of age and sex on mechanisms of cytoprotection (autophagy and the HSR) and cellular stress (apoptotic signaling and the acute inflammatory response) during ex vivo hypothermic cooling. Venous blood samples from 20 healthy young (10 females; mean [SD]: 22 [2] years) and 20 healthy older (10 females; 66 [5] years) adults were either isolated immediately (baseline) for peripheral blood mononuclear cells (PBMCs) or exposed to water bath temperatures maintained at 37, 35, 33, 31, or 4 °C for 90 min before PBMC isolation. Proteins associated with autophagy, apoptosis, the HSR, and inflammation were analyzed via Western blotting. Indicators of autophagic initiation and signaling (LC3, ULK1, and beclin-2) and the HSR (HSP90 and HSP70) increased when exposed to hypothermic temperatures in young and older adults (all p ≤ 0.007). Sex-related differences were only observed with autophagic initiation (ULK1; p = 0.015). However, despite increases in autophagic initiators ULK1 and beclin-2 (all p < 0.001), this was paralleled by autophagic dysfunction (increased p62) in all groups (all p < 0.001). Further, apoptotic (cleaved-caspase-3) and inflammatory (IL-6 and TNF-α) signaling increased in all groups (all p < 0.001). We demonstrated that exposure to hypothermic conditions is associated with autophagic dysfunction, irrespective of age or sex, although there may exist innate sex-related differences in cytoprotection in response to cold exposure as evidenced through altered autophagic initiation.

Involvement of nitric oxide synthase and reactive oxygen species in TRPA1-mediated cutaneous vasodilation in young and older adults.

OBJECTIVE: To investigate the nitric oxide synthase (NOS) and reactive oxygen species (ROS) contributions of the cutaneous vasodilator response to transient receptor potential ankyrin-1 channel (TRPA1) activation in young and older adults. MATERIALS AND METHODS: In sixteen young (20 ± 2 years, 8 females) and sixteen older adults (61 ± 5 years, 8 females), cutaneous vascular conductance normalized to maximum vasodilation (%CVCmax) was assessed at four dorsal forearm skin sites continuously perfused via microdialysis with: 1) vehicle solution (Control, 2 % dimethyl sulfoxide, 2 % Ringer, 96 % propylene glycol), 2) 10 mM Ascorbate (non-specific ROS inhibitor), 3) 10 mM L-NAME (non-specific NOS inhibitor), or 4) Ascorbate+L-NAME. The TRPA1 agonist cinnamaldehyde was co-administered at all sites [0 % (baseline), 2.9 %, 8.8 %, 26.4 %; ≥ 30 min per dose]. RESULTS: %CVCmax was not different between groups for Control, L-NAME, and Ascorbate (all p > 0.05). However, there were significant main dose effects for each site wherein %CVCmax was greater than baseline from 2.9 % to 26.4 % cinnamaldehyde for Control and Ascorbate, and at 26.4 % cinnamaldehyde for L-NAME and Ascorbate+L-NAME (all p < 0.05). For Ascorbate+L-NAME, there was a significant main group effect, wherein perfusion was 6 %CVCmax [95% CI: 2, 11, p < 0.05] greater in the older compared to the young group across all cinnamaldehyde doses. There was a significant main site effect for area under the curve wherein L-NAME and Ascorbate+L-NAME were lower than Control and Ascorbate across groups (all p < 0.05). CONCLUSION: The NOS-dependent cutaneous vasodilator response to TRPA1 activation is maintained in older adults, with no detectable contribution of ascorbate-sensitive ROS in either age group.

Serum klotho concentrations in older men with hypertension or type 2 diabetes during prolonged exercise in temperate and hot conditions.

PURPOSE: Klotho is a cytoprotective protein that increases during acute physiological stressors (e.g., exercise heat stress), although age-related declines in klotho may underlie cellular vulnerability to heat stress. The present study aimed to compare serum klotho in healthy older men and men with type 2 diabetes (T2D) or hypertension (HTN) during prolonged exercise in temperate or hot conditions. METHODS: We evaluated serum klotho in 12 healthy older men (mean [SD]; 59 years [4]), 10 men with HTN (60 years [4]), and 9 men with T2D (60 years [5]) before and after 180 min of moderate-intensity (fixed metabolic rate of 200 W/m2; ~ 3.4 METs) exercise and 60 min of recovery in temperate (wet-bulb globe temperature (WBGT) 16 °C) and hot (WBGT 32 °C) environments. Core temperature (rectal), heart rate (HR), and heart rate reserve (HRR) were measured continuously while klotho was measured at the end of baseline, exercise, and recovery. RESULTS: Total exercise duration was reduced during the hot condition in older men with HTN and T2D than healthy older men (both p ≤ 0.049), despite similar core temperatures, HR, and HRR. Klotho was higher than rest following exercise in the heat in healthy older men (+ 191 pg/mL [189]; p < 0.001) and responses were greater (p = 0.036) than men with HTN (+ 118 pg/mL [49]; p = 0.030), although klotho did not increase in men with T2D (+ 4 pg/mL [71]; p ≥ 0.638). CONCLUSION: Given klotho's role in cytoprotection, older men with HTN and especially T2D may be at increased cellular vulnerability to prolonged exercise or physically demanding exercise in the heat.

Impacts of age, diabetes, and hypertension on serum endothelial monocyte-activating polypeptide-II after prolonged work in the heat.

BACKGROUND: With rising temperature extremes, older workers are becoming increasingly vulnerable to heat-related injuries because of age- and disease-associated decrements in thermoregulatory function. Endothelial monocyte-activating polypeptide-II (EMAP-II) is a proinflammatory cytokine that has not yet been well-characterized during heat stress, and which may mediate the inflammatory response to high levels of physiological strain. METHODS: We evaluated serum EMAP-II concentrations before and after 180 min of moderate-intensity work (200 W/m2 ) in temperate (wet-bulb globe temperature [WBGT] 16°C) and hot (WBGT 32°C) environments in heat-unacclimatized, healthy young (n = 13; mean [SD]; 22 [3] years) and older men (n = 12; 59 [4] years), and unacclimatized older men with hypertension (HTN) (n = 10; 60 [4] years) or type 2 diabetes (T2D) (n = 9; 60 [5] years). Core temperature and heart rate were measured continuously. RESULTS: In the hot environment, work tolerance time was lower in older men with HTN and T2D compared to healthy older men (both p < 0.049). While core temperature and heart rate reserve increased significantly (p < 0.001), they did not differ across groups. End-exercise serum EMAP-II concentrations were higher in young men relative to their older counterparts due to higher baseline levels (both p ≤ 0.02). Elevations in serum EMAP-II concentrations were similar between healthy older men and older men with HTN, while serum EMAP-II concentrations did not change in older men with T2D following prolonged work in the heat. CONCLUSION: Serum EMAP-II concentrations increased following prolonged moderate-intensity work in the heat and this response is influenced by age and the presence of HTN or T2D.

Influence of uncomplicated, controlled hypertension on local heat-induced vasodilation in nonglabrous skin across the body.

The objective of this study was 1) to examine pooled effects of hypertension on nitric oxide (NO)-dependent vasodilation during local heating across multiple nonglabrous skin regions, and 2) explore regional differences. Responses were compared between 14 participants with uncomplicated hypertension controlled with medication (7 females, 61 ± 6 yr) and 14 age-matched nonhypertensive controls (6 females; 60 ± 5 yr). Cutaneous vascular conductance, normalized to maximum vasodilation (%CVCmax), was assessed at the upper chest, abdomen, dorsal forearm, thigh, and lateral calf during local heating. Across all regions, local skin temperatures were simultaneously increased from 33°C to 42°C (1°C·10 s-1) and held until a stable heating plateau was achieved (∼40 min), followed by continuous infusion of 20 mM of NG-nitro-l-arginine methyl ester (l-NAME; ∼40 min) at all sites until a stable l-NAME plateau was achieved. The difference between heating and l-NAME plateaus was defined as the NO-contribution. Statistical equivalence for each heating phase was determined based on equivalence bounds of ±10%CVCmax for between-group differences. Pooled (all-regions) %CVCmax responses were equivalent for baseline (two one-sided t tests; P < 0.001), heating plateau (P = 0.002), l-NAME plateau (P = 0.028), and NO-contribution (P = 0.003). For individual regions, responses were equivalent at baseline for the abdomen, thigh, and calf, the heating plateau for the thigh, and the l-NAME plateau for the calf (all P < 0.05). Conversely, the calf heating plateau was lower in the hypertension group (t test; P < 0.05). Local heat-induced cutaneous vasodilation was statistically equivalent between individuals with uncomplicated, controlled hypertension, and nonhypertensive age-matched adults when pooled across multiple skin sites. Conversely, individual between-region comparisons were generally too variable to permit definitive conclusions.

Autophagic response to exercise in peripheral blood mononuclear cells from young men is intensity-dependent and is altered by exposure to environmental heat.

Autophagy is essential to maintaining cellular homeostasis in all eukaryotic cells and to tolerance of acute stressors such as starvation, heat, and recovery after exercise. Limited information exists regarding the exercise intensity-dependent autophagic response in humans, and it is unknown how environmental heat stress may modulate this response. Therefore, we evaluated autophagy and accompanying pathways of cellular stress [the heat-shock response (HSR), apoptosis, and acute inflammation] in peripheral blood mononuclear cells (PBMCs) from 10 young men (mean [SD]; 22 [2] years) before, immediately after and up to 6-h postexercise recovery from 30 min of low-, moderate-, and high-intensity semirecumbent cycling [40%, 55%, and 70% of maximal oxygen consumption (V̇o2max), respectively] in a temperate environment (25°C) and at 70% of V̇o2max in a hot environment (40°C). Changes in protein content were analyzed via Western blot. Each increase in exercise intensity was associated with elevations in mean body temperature. LC3-II increased after moderate-intensity exercise, with further increases after high-intensity exercise (P < 0.05). However, an increase in beclin-2 and ULK1, with a decrease in p62 was only observed after high-intensity exercise, which was paralleled by elevated TNF-α and cleaved-caspase-3, with the HSR peaking at 6 h after exercise (P < 0.05). When exercise was performed in the heat, greater LC3-II and cleaved-caspase-3 accumulation were observed; however, beclin-2 declined in recovery (P < 0.05). Therefore, our findings indicate that autophagy in PBMCs during exercise may be associated with greater heat strain exhibited during increasing exercise intensities, which is modulated by exposure to heat.

The effect of acute intradermal administration of ascorbate on heat loss responses in older adults with uncomplicated controlled hypertension.

NEW FINDINGS: What is the central question of this study? Does acute intradermal administration of the antioxidant ascorbate augment local forearm cutaneous vasodilatation and sweating via nitric oxide synthase (NOS)-dependent mechanisms during exercise-heat stress in older adults with uncomplicated controlled hypertension? What is the main finding and its importance? Relative to the control site, ascorbate had no effect on forearm cutaneous vascular conductance (CVC) and sweat rate, although CVC was reduced with NOS inhibition in older adults with hypertension. Acute local administration of ascorbate to forearm skin does not modulate heat loss responses during exercise-heat stress in older adults with hypertension. ABSTRACT: Nitric oxide synthase (NOS) contributes to the heat loss responses of cutaneous vasodilatation and sweating during exercise. However, the contribution of NOS may be attenuated in individuals with uncomplicated, controlled hypertension due to elevated oxidative stress, which can reduce NO bioavailability. We evaluated the hypothesis that the acute local intradermal administration of the antioxidant ascorbate would enhance cutaneous vasodilatation and sweating via NOS-dependent mechanisms during an exercise-heat stress in adults with hypertension. Habitually active adults who were normotensive (n = 14, 7 females, 62 ± 4 years) or had uncomplicated, controlled hypertension (n = 13, 6 females, 62 ± 5 years) performed 30 min of moderate-intensity (50% of their pre-determined peak oxygen uptake) semi-recumbent cycling in the heat (35°C, 20% relative humidity). Cutaneous vascular conductance (CVC) and sweat rate were assessed at four forearm skin sites continuously perfused with (1) lactated Ringer solution (Control), (2) 10 mM antioxidant ascorbate, (3) 10 mM NG -nitro-l-arginine methyl ester (l-NAME), a non-selective NOS inhibitor, or (4) a combination of ascorbate and l-NAME. Relative to Control, no effect of ascorbate was observed on CVC or sweating in either group (P = 0.619). However, l-NAME reduced CVC relative to Control in both groups (P ≤ 0.038). No effect of any treatment on sweating was observed (P ≥ 0.306). Thus, acute local administration of ascorbate to forearm skin does not enhance the activation of heat loss responses of cutaneous vasodilatation and sweating in older adults, and those with hypertension during an exercise-heat stress.

Exercise in the heat induces similar elevations in serum irisin in young and older men despite lower resting irisin concentrations in older adults.

Irisin is thought to play a cytoprotective role during acute stressors, such as exercise, by reducing oxidative stress and inflammation. Relative to young adults, older individuals exhibit an impaired capacity to dissipate heat during exercise, which can exacerbate elevations in oxidative stress and the acute inflammatory response especially in the heat. In turn, this could induce a greater increase in circulating irisin. Thus, we evaluated age-related differences in irisin expression during prolonged exercise in a non-heat stress and high-heat stress environment. Specifically, we assessed serum irisin in 12 young (22 ± 3 years) and 12 older (59 ± 4 years) men before and after 3-h moderate-intensity exercise (metabolic rate: 200 W/m2) and 60-min post-exercise recovery in temperate (wet-bulb globe temperature (WBGT) 16 °C) and hot (WBGT 32 °C) environments. Core temperature (Tco) was measured continuously. Post-exercise Tco was similarly higher in the hot compared to the temperate condition for both groups (p < 0.001), although Tco remained elevated at end-recovery in the heat in older but not young adults (p = 0.006). Absolute serum irisin concentrations were significantly higher (p ≤ 0.002) under all conditions in the young relative to older adults. Post-exercise and end-recovery irisin was elevated above baseline in both groups in the hot (+39.3 pg/mL SEM 8 and + 48.9 pg/mL SEM 10, respectively; both p ≤ 0.043) but not the temperate condition. When comparing between conditions, the change in irisin concentrations at post-exercise did not differ, although serum irisin was elevated in the hot (+48.9 pg/mL SEM 10) relative to the temperate (+0.88 pg/mL SEM 0.2) condition in both groups at end-recovery (p = 0.004). Our findings indicate that irisin concentrations were elevated after exercise compared to rest in hot, but not temperate conditions across groups. However, older adults may still have greater cellular vulnerability to heat stress given their blunted circulating irisin levels.

The serum irisin response to prolonged physical activity in temperate and hot environments in older men with hypertension or type 2 diabetes.

Current labor demographics are changing, with the number of older adults increasingly engaged in physically demanding occupations expected to continually rise, which are often performed in the heat. Given an age-related decline in whole-body heat loss, older adults are at an elevated risk of developing heat injuries that may be exacerbated by hypertension (HTN) and type 2 diabetes (T2D). Elevated irisin production may play a role in mitigating the excess oxidative stress and acute inflammation associated with physically demanding work in the heat. However, the effects of HTN and T2D on this response remain unclear. Therefore, we evaluated serum irisin before and after 3-h of moderate intensity exercise (metabolic rate: 200 W/m2) and at the end of 60-min of post-exercise recovery in a temperate (wet-bulb globe temperature (WBGT) 16 °C) and high-heat stress (WBGT 32 °C) environment in 12 healthy older men (mean ± SD; 59 ± 4 years), 10 men with HTN (60 ± 4 years), and 9 men with T2D (60 ± 5 years). Core temperature (Tco) was measured continuously. In the heat, total exercise duration was significantly lower in older men with HTN and T2D (both, p ≤ 0.049). Despite Tco not being different between groups, Tco was higher in the hot compared to the temperate condition for all groups (p < 0.001). Similarly, serum irisin concentrations did not differ between groups under either condition but were elevated relative to the temperate condition during post-exercise and end-recovery in the heat (+93.9 pg/mL SEM 26 and + 70.5 pg/mL SEM 38 respectively; both p ≤ 0.014). Thus, our findings indicate similar irisin responses in HTN and T2D compared to healthy, age-matched controls, despite reduced exercise tolerance during prolonged exercise in the heat. Therefore, older workers with HTN and T2D may exhibit greater cellular stress during prolonged exercise in the heat, underlying greater vulnerability to heat-induced cellular injury.

Regional variation in nitric oxide-dependent cutaneous vasodilatation during local heating in young adults.

NEW FINDINGS: What is the central question of this study? Are regional differences in nitric oxide (NO)-dependent cutaneous vasodilatation during local skin heating present in young adults? What is the main finding and its importance? NO-dependent cutaneous vasodilatation varied across the body. The abdomen demonstrated larger NO contributions, while the chest demonstrated smaller NO contributions, compared to other regions. This exploratory work is an important first step in characterizing regional heterogeneity of cutaneous microvascular control across the torso and limbs. Equally, it serves to generate hypotheses for future studies examining regional cutaneous microvascular control in ageing and disease. ABSTRACT: Regional variations in cutaneous vasodilatation during local skin heating exist across the body. While nitric oxide (NO) is a well-known modulator of this response, the extent of regional differences in NO-dependent cutaneous vasodilatation during local skin heating remains uncertain. In 16 habitually active young adults (8 females; 25 ± 5 years), cutaneous vascular conductance, normalized to maximum vasodilatation (% CVCmax ), was assessed at the upper chest, abdomen, dorsal forearm, thigh and lateral calf during local skin heating. Across all regions, local skin temperatures were simultaneously increased from 33 to 42°C (1°C per 10 s), and held until a stable heating plateau was achieved (∼40 min). Next, with local skin temperature maintained at 42°C, 20 mM of NG -nitro-l-arginine methyl ester (l-NAME) was continuously infused at each site until a stable l-NAME plateau was achieved (∼40 min). The difference between heating and l-NAME plateaus was identified as the NO contribution for each region. There was no evidence for region-specific responses at baseline (P = 0.561), the heating plateau (P = 0.351) or l-NAME plateau (P = 0.082), but there was for the NO contribution (P = 0.048). Overall, point estimates for between-region differences in the NO contribution varied across the body from 0 to 19% CVCmax . The greatest effects were observed for the abdomen, wherein the NO contribution was consistently greater than for the other regions (range: 9-19% CVCmax ). The chest was consistently lower than the other regions (range: 7-19% CVCmax ). The smallest effects were observed between limb regions (range: 0-2% CVCmax ). These findings advance our understanding of the mechanisms influencing regional variations in the cutaneous vasodilator response to local skin heating in young adults.

Impaired autophagy following ex vivo heating at physiologically relevant temperatures in peripheral blood mononuclear cells from elderly adults.

With the increasing threat of climate change and the accompanying rise in the frequency and severity of extreme heat events, there are growing health concerns for heat-vulnerable elderly adults. Elderly adults are at increased risk of developing heat-related injuries, in part due to age-related declines in thermoregulatory and cellular function. Regarding the latter, the process of autophagy is activated as a cellular protective mechanism to counter heat-induced stress, but the extent that heat stress activates autophagy in elderly adults is not known. Further, the interplay between autophagy, the heat shock response (HSR), the acute inflammatory response, and apoptosis remains poorly understood in elderly adults. Therefore, the purpose of this study was to examine changes in autophagy, the HSR, inflammation, and apoptosis following increasing levels of ex vivo heat stress representative of physiologically relevant increases in body core temperatures (37-41 °C). Whole blood from 20 elderly adults (72 ± 4 years; 14 men, 6 women) was heated (via water immersion) to temperatures representative of normal resting conditions (normothermia; 37 °C), in addition to moderate and severe heat stress conditions (39, and 41 °C, respectively) for 90 min. Peripheral blood mononuclear cells (PBMC) were isolated and protein markers of autophagy, the HSR, acute inflammation, and apoptosis were examined. No significant increases in markers of autophagy or the HSR were observed following any temperature condition. However, an increase in acute inflammation was observed above baseline following moderate heat stress (39 °C), with further increases in inflammation and apoptosis observed during severe heat stress (41 °C). Our findings indicate that PBMCs from elderly adults do not exhibit increases in autophagy or the HSR following severe heat stress, potentially contributing to the elevated risk of cellular dysfunction seen in elderly adults during heat stress.

Regulation of autophagy following ex vivo heating in peripheral blood mononuclear cells from young adults.

Under conditions of extreme heat stress, the process of autophagy has previously been shown to protect human cells, but the exact body temperature at which autophagic activation occurs is largely unknown. Further, the interplay between autophagy, the heat shock response (HSR), inflammation, and apoptosis have yet to be examined together under temperature conditions representative of human internal body temperatures at rest (37 °C) or under severe heat stress conditions (41 °C). Thus, the purpose of this study was to examine threshold changes in autophagy, the HSR, inflammation, and apoptosis to increasing levels of ex vivo heat stress. Whole blood was collected from 20 young (23 ± 4 years; 10 men, 10 women) physically active participants. Peripheral blood mononuclear cells (PBMCs) were isolated immediately (baseline) and after 90-min of whole blood heating in 37, 39, and 41 °C water baths, representative of normal resting (non-heat stress) as well as moderate and severe heat stress conditions in humans, respectively. At 37 °C, increased autophagic activity was demonstrated, with no change in the HSR, and inflammation. Subsequently, responses of autophagy, the HSR, and inflammation increased with a moderate heat stress (39 °C), with further increases in only autophagy and the HSR under a severe heat stress of 41 °C. We observed no increase in apoptosis under any temperature condition. Our findings show that in human PBMCs, the autophagy and HSR systems may act cooperatively to suppress apoptotic signaling following heat stress, which may in part be mediated by an acute inflammatory response.

Temperature-Dependent Relationship of Autophagy and Apoptotic Signaling During Cold-Water Immersion in Young and Older Males.

Autophagy is a crucial cytoprotective mechanism preventing the accumulation of cellular damage, especially during external stimuli such as cold exposure. Older adults poorly tolerate cold exposure and age-related impairments in autophagy may contribute to the associated reductions in cold tolerance. The purpose of this investigation is to evaluate the effect of different intensities of in vivo cold-water immersion and in vitro cold exposure on autophagic and apoptotic signaling in young and older males. Peripheral blood mononuclear cells (PBMCs) are isolated at baseline, end-cold exposure, and after 3 h of thermoneutral recovery. Additionally, PBMCs are treated with rapamycin and bafilomycin prior to in vitro cold exposure equivalent to in vivo core temperatures (35-37 °C). Proteins associated with autophagy, apoptosis, the heat shock response, and inflammation are analyzed via Western blotting. Moderate cold stress (0.5 °C decrease in core temperature) increased autophagic and heat shock protein activity while high cold stress (1.0 °C decrease in core temperature) augmented apoptosis in young males. In older males, minimal autophagic activation during both cold-water exposures are associated with increased apoptotic and inflammatory proteins. Although in vitro cold exposure confirmed age-related dysfunction in autophagy, rapamycin-induced stimulation of autophagic proteins underlie the potential to reverse age-related vulnerability to cold exposure.

Markers of enterocyte damage, microbial translocation, and systemic inflammation following 9 h of heat exposure in young and older adults.

Heat stress induced damage to the gastrointestinal barrier can induce local and systemic inflammatory reactions implicated in heat-stroke. Gastrointestinal barrier damage has been shown to be greater in older relative to young adults following hyperthermia. However, comparisons between young and older adults have been limited to brief exposures (3 h), which may not reflect the duration of heat stress experienced during heat waves. We therefore evaluated markers of intestinal epithelial damage (log transformed intestinal fatty acid binding protein, IFABPLOG), microbial translocation (soluble cluster of differentiation 14, sCD14LOG), and systemic inflammation (tumour necrosis factor alpha, TNF-αLOG; interleukin 6, IL-6LOG; C-reactive protein, CRP) in 19 young (interquartile range: 21-27 years; 10 females) and 37 older (68-73 years; 10 females) adults before and after 9 h of rest in 40 °C (9% relative humidity). The magnitude of the increase in IFABPLOG was 0.38 log pg/mL (95% CI, 0.10, 0.65 log pg/mL) greater in the older relative to young cohort (P = 0.049) after 9 h heat exposure. At baseline both IL-6LOG and CRP concentrations were higher in the older (IL-6: 2.67 (1.5) log pg/mL, CRP: 0.28 (1.5) mg/mL) relative to the young (IL-6: 1.59 log pg/mL, SD 1.2; CRP: 0.11 mg/mL, SD 1.7) group (both P ≤ 0.001). The change in IL-6 and CRP was similar between groups following 9 h heat exposure (IL-6: P = 0.053; CRP: P = 0.241). Neither sCD14LOG and TNF-αLOG were different between groups at baseline nor altered after 9 h heat exposure. Our data indicate that age may modify intestinal epithelial injury following 9 h of passive heat exposure.

Effect of daylong exposure to indoor overheating on autophagy and the cellular stress response in older adults.

To protect vulnerable populations during heat waves, public health agencies recommend maintaining indoor air temperature below ∼24-28 °C. While we recently demonstrated that maintaining indoor temperatures ≤26 °C mitigates the development of hyperthermia and cardiovascular strain in older adults, the cellular consequences of prolonged indoor heat stress are poorly understood. We therefore evaluated the cellular stress response in 16 adults (six females) aged 66-78 years during 8 h rest in ambient conditions simulating homes maintained at 22 °C (control) and 26 °C (indoor temperature upper limit proposed by health agencies), as well as non-air-conditioned domiciles during hot weather and heat waves (31 and 36 °C, respectively; all 45% relative humidity). Western blot analysis was used to assess changes in proteins associated with the cellular stress response (autophagy, apoptosis, acute inflammation, and heat shock proteins) in peripheral blood mononuclear cells harvested prior to and following exposure. Following 8 h exposure, no cellular stress response-related proteins differed significantly between the 26 and 22 °C conditions (all, P ≥ 0.056). By contrast, autophagy-related proteins were elevated following exposure to 31 °C (p62: 1.5-fold; P = 0.003) and 36 °C (LC3-II, LC3-II/I, p62; all ≥2.0-fold; P ≤ 0.002) compared to 22 °C. These responses were accompanied by elevations in apoptotic signaling in the 31 and 36 °C conditions (cleaved-caspase-3: 1.8-fold and 3.7-fold, respectively; P ≤ 0.002). Furthermore, HSP90 was significantly reduced in the 36 °C compared to 22 °C condition (0.7-fold; P = 0.014). Our findings show that older adults experience considerable cellular stress during prolonged exposure to elevated ambient temperatures and support recommendations to maintain indoor temperatures ≤26 °C to prevent physiological strain in heat-vulnerable persons.

Greater hyperthermia in men with type 2 diabetes does not lead to higher serum levels of cellular stress biomarkers following exercise-heat stress.

Type 2 diabetes (T2D) is associated with worsening age-related impairments in heat loss, causing higher core temperature during exercise. We evaluated whether these thermoregulatory impairments occur with altered serum protein responses to heat stress by measuring cytoprotection, inflammation, and tissue damage biomarkers in middle-aged-to-older men (50-74 years) with (n = 16) and without (n = 14) T2D following exercise in 40°C. There were no changes in irisin, klotho, HSP70, sCD14, TNF-α, and IL-6, whereas NGAL (+539 pg/mL, p = 0.002) and iFABP (+250 pg/mL, p < 0.001) increased similarly across groups. These similar response patterns occurred despite elevated core temperature in individuals with T2D, suggesting greater heat vulnerability.

Physiological responses to 9 hours of heat exposure in young and older adults. Part III: Association with self-reported symptoms and mood state.

Older adults are at greater risk of heat-related morbidity and mortality during heat waves, which is commonly linked to impaired thermoregulation. However, little is known about the influence of increasing age on the relation between thermal strain and perceptual responses during daylong heat exposure. We evaluated thermal and perceptual responses in 20 young (19-31 yr) and 39 older adults (20 with hypertension and/or type 2 diabetes; 61-78 yr) resting in the heat for 9 h (heat index: 37°C). Body core and mean skin temperature areas under the curve (AUC, hours 0-9) were assessed as indicators of cumulative thermal strain. Self-reported symptoms (68-item environmental symptoms questionnaire) and mood disturbance (40-item profile of mood states questionnaire) were assessed at end-heating (adjusted for prescores). Body core temperature AUC was 2.4°C·h [1.0, 3.7] higher in older relative to young adults (P < 0.001), whereas mean skin temperature AUC was not different (-0.5°C·h [-4.1, 3.2] P = 0.799). At end-heating, self-reported symptoms were not different between age groups (0.99-fold [0.80, 1.23], P = 0.923), with or without adjustment for body core or mean skin temperature AUC (both P ≥ 0.824). Mood disturbance was 0.93-fold [0.88, 0.99] lower in older, relative to young adults (P = 0.031). Older adults with and without chronic health conditions experienced similar thermal strain, yet those with these conditions reported lower symptom scores and mood disturbance compared with young adults and their age-matched counterparts (all P ≤ 0.026). Although older adults experienced heightened thermal strain during the 9-h heat exposure, they did not experience greater self-reported symptoms or mood disturbance relative to young adults.NEW & NOTEWORTHY Despite experiencing greater cumulative thermal strain during 9 h of passive heat exposure, older adults reported similar heat-related symptoms and lower mood disturbance than young adults. Furthermore, self-reported symptoms and mood disturbance were lower in older adults with common age-associated health conditions than young adults and healthy age-matched counterparts. Perceptual responses to heat in older adults can underestimate their level of thermal strain compared with young adults, which may contribute to their increased heat vulnerability.

Brief ambient cooling preserves autophagy in peripheral blood mononuclear cells from older adults during 9 h of heat exposure.

Heat waves can cause dangerous elevations in body temperature that can compromise cellular function and increase the risk of heat stroke and major cardiovascular events. Visiting a cooling center or other air-conditioned location is commonly recommended by health agencies to protect heat-vulnerable older persons but the associated cellular effects remain underexplored. We evaluated cellular stress responses in peripheral blood mononuclear cells (PBMC) from 19 older adults [71 (SD 2) yr; 9 females] before and after a 9-h heat exposure [40.3°C and 9.3% relative humidity (RH)], with participants moved to a cool room (∼23°C) for hours 5 and 6 (cooling group). Responses were compared with 17 older adults [72 (4) yr; 7 females] who remained in the heat for the entire 9 h (control group). Changes in proteins associated with autophagy, apoptotic signaling, acute inflammation, and the heat shock response (HSR) were assessed via Western blot. Although both groups experienced similar elevations in physiological strain before the cooling center intervention, brief cooling resulted in stark albeit transient reductions in core temperature and heart rate. At end-exposure, autophagy proteins LC3-II and p62 were elevated 1.9-fold [95% CI: 1.2, 2.8] and 2.3-fold [1.4, 3.8], respectively, in the control group relative to cooling group. This was paired with a 2.8-fold [1.6, 4.7] greater rise in apoptotic protein cleaved-caspase-3 in the control group compared with the cooling group. Our findings indicate that 2 h of ambient cooling midway through a 9-h simulated heat wave may preserve autophagy and mitigate heat-induced cellular stress in older adults.NEW & NOTEWORTHY Heat waves can lead to dangerous elevations in body temperature, increasing the risk of life-threatening health conditions. Visiting a cooling center or other air-conditioned location is commonly recommended to protect heat-vulnerable older persons, although the effects on the cellular stress response remain unknown. We found that 2 h of ambient cooling midway through a 9 h simulated heat wave preserves autophagy, a vital cellular survival mechanism, and mitigates accompanying pathways of cellular stress in older adults.