Monocyte heat shock protein 72 at rest and in response to environmental and exercise stress : implications for cross tolerance in vivo

Taylor, Lee

Abstract

The human body endures stress on a daily basis, with many occupational and recreational activities beset with such challenges to homeostasis. These challenges include that of exercise and exposure to challenging environments (hypoxia and hyperbaria). A group of specialised proteins, termed heat shock proteins (HSP) provide protection to such stressors at a cellular level. This cellular defence mechanism protects and oversees whole body protein homeostasis, which is vital to all cellular processes.

One such protective HSP, is HSP72, which is present in almost all cellular compartments and has received extensive and widespread research interest – with elevations in HSP72 indicatively linked to augmented cellular and whole body resistance to various exercise and environmental stressors. Despite this extensive research interest, several fundamental areas of concern with regard to HSP72 have not been satisfactorily addressed or delineated. In general, the experimental chapters of this thesis were designed to investigate several broad research questions related to those areas that have not been sufficiently addressed, as highlighted by the Literature Review. These areas include the reliance on thermal and/or mechanical stress to induce elevations in HSP72, both in vitro and in vivo, to initiate conveyed cellular protection. No in vivo attempts have been made to use a non-thermal and/or non-mechanical based stimulus, such as a hypoxic or hyperbaric exposure, to induce elevations in basal HSP72 in an attempt to confer cellular tolerance to future episodes of stress. Additionally, any potential relationships between changes in redox balance and stress induced changes in HSP72 expression have not been investigated in vivo, this potential interplay could be important when discussing any likely mechanisms for HSP72 dependent conferred cellular tolerance. In order to investigate such hypoxic or hyperbaric mediated changes in basal HSP72 expression securely, basal expression of monocyte expressed HSP72 (mHSP72) warrants investigation (diurnal and/or circadian variation), as, at present, this has not be conducted securely or adequately.

The first experimental chapter investigated basal expression of mHSP72 over a 24 h period. Seventeen recreationally active (mean ± SD: 5.9±2.2 h∙wk-1) male subjects (19.8±4.3 yr, 177±6.4 cm, 75.7±10.9 kg) had blood samples taken every 4 h from 0900 until 0900 the next day, at rest, within a temperature regulated laboratory. Core temperature, as assessed by ingestible telemetric temperature sensor pill, was obtained at 5 min intervals. Basal mHSP72 expression was found to follow a circadian rhythm, which was correlated to core temperature (rs=0.41, p<0.001). Notably, during “waking” hours (0900 – 2100), this circadian rhythm was shown to follow a quadratic trend in expression (F = 21.2, p < 0.001).

The second experimental chapter investigated the repeatability of the quadratic trend in basal mHSP72 expression demonstrated within the previous experimental chapter. Twelve healthy recreationally active (mean ± SD: 5.2±1.9 h∙wk-1) male subjects (20.2±1.9 yr, 178.7±5.6 cm, 75.1±6.0 kg) had blood samples taken on three separate days (separated by three days) over a 9 h period (0800, 1100, 1400) at rest within a temperature regulated laboratory. Results supported those from the previous chapter, whereby, the quadratic trend in basal mHSP72 expression was evident on three separate days (F = 26.0; p = 0.001; partial η2 = 0.74), where mHSP72 decreased between 0800 and 1100 (mean difference = -17%; 95% CI = -24%, -10%; p < 0.001) and then increased between 1100 and 1400 (mean difference = 8%; 95% CI = 2%, 14%; p = 0.015). In conjunction with the first experimental chapter, these results demonstrate the importance in controlling the time of day interventions are administered in vivo, as differential responses may be seen due to differences in basal HSP72 expression. Furthermore, when regular blood samples are required post intervention, the timetabling of such collections needs to be stringently adhered to, due to within-day variation in basal mHSP72. Differing basal values of mHSP72 are known to determine the magnitude of post stressor mHSP72 expression and thus any variation (even minimal) in basal mHSP72 is important.

The third experimental chapter investigated the potential of an environmental stressor to disrupt the quadratic trend in basal mHSP72 and explored whether any such changes in mHSP72 may have a relationship with alterations in redox balance. Six healthy recreationally active (mean ± SD: 5.9±2.3 h.wk-1) male subjects (mean ± SD: 21.3±7.2 yr, 179.2±4.8 cm, 79.3±9.9 kg) participated within the study. Control values (NA) for mHSP72 were obtained one week before the first hyperbaric air (HA) exposure with the hyperbaric oxygen (HBO) exposure following a week later (i.e. 3 study days NA, HA and HBO each separated by one week). These exposures commenced at 1500 and involved a simulated dive consisting of HA (2.8 ATA) or HBO (20 min O2, 5 min HA cycle) within a hyperbaric chamber constituting 78 min bottom time. Within each study day blood samples were taken at 0900, 1300, 1700 and 2100. The administration of HBO and HA were sufficient to disrupt the quadratic trend shown within the NA condition (F = 27.6, p < 0.001). The model demonstrated significant main effects for condition (F = 24.7, p < 0.001) and time (F = 9.6, p < 0.001), and a condition x time interaction effect was also observed (F = 7.1, p < 0.001). Decomposition of this interaction effect revealed a reduction in mHSP72 was evident post hyperbaric exposures, whereby, mHSP72 expression at 1700 was significantly higher in NA than in HA (p = 0.016) and HBO (p < 0.001), this reduction was still evident in both HA and HBO compared to NA at 2100 (p < 0.001). In addition to quantification of mHSP72, a measure of oxidative stress, thiobarbituric acid reactive substances (plasma TBARS), was also retrospectively assessed from the isolated plasma of these blood samples. There were no significant main effects observed for condition (F = 0.7; p = 0.50) or time (F = 0.06; p = 0.81), and no significant condition x time interaction effect (F = 0.5; p = 0.62) for plasma TBARS. Despite the failure of the hyperbaric environments to elicit increases in basal mHSP72, one important physiological contribution may be contrived of this reduction in mHSP72, as in vitro and in vivo low basal mHSP72 content is indicatively correlated to enhanced post stressor HSP72 expression. Such hyperbaric mediated reductions in basal content may allow enhanced HSP72 expression post stressor, an intervention which may be of benefit to hyperthermic exercise acclimation protocols which seek elevated mHSP72 as part of the in vivo heat acclimation process.

The fourth experimental chapter employed an acute hypoxic exposure (75 min, 2980 m) at rest in an attempt to disrupt the previously demonstrated quadratic trend in basal mHSP72 expression and explored whether any such changes in mHSP72 may have a relationship with alterations in redox balance. Twelve healthy recreationally active (mean ± SD: 5.1±1.5 h.wk-1) male subjects (19.8±3.5 yr, 175.5±10.8 cm, 73.1±8.0 kg) participated in the study. Testing was conducted on consecutive days, with all subjects providing control samples on this first day with the hypoxic exposure administered on the second day. This exposure commenced and ceased at 0930 and 1045 respectively. Blood samples were taken at 0800, 1100, 1400, 1700 and 2000. In addition to quantification of mHSP72 a measure of oxidative stress, plasma TBARS, was also retrospectively assessed from the isolated plasma of these blood samples. There was a significant quadratic trend in mHSP72 for the control condition (F = 23.5; p = 0.002; partial η2 = 0.77) with no such trend evident for the hypoxic condition (largest F ratio was for a quadratic trend: F = 3.9; p = 0.087). The condition-by-time interaction was significant for mHSP72 (F = 19.5; p = 0.003; partial η2 = 0.74), where the difference between the control and hypoxic conditions were significantly different at 1100 (p = 0.002), 1400 (p < 0.001), 1700 (p = 0.034) and 2000 (p = 0.041). No significant trend was observed for plasma TBARS in the control condition (F = 0.8; p = 0.41), but a significant quadratic trend was evident for the hypoxia condition (F = 36.1; p = 0.001; η2 = 0.84). Plasma TBARS increased by 98% (95% CI 30%, 166%) from pre-intervention to immediately post-intervention and decreased thereafter until pre-intervention levels were reached at 2000. This difference in trends within the plasma TBARS data between conditions showed up as a linear-by-quadratic interaction in the two-way analysis (F = 41.5; p < 0.001; η2 = 0.86), where significant differences between conditions were observed only at 1100 (p = 0.006) and 1400 (p = 0.032). The results demonstrate that an acute hypoxic exposure (75 min, 2980 m) was sufficient to induce significant increases in mHSP72 post intervention. This increase in mHSP72 may be linked to the significant increase in oxidative stress (plasma lipid peroxidation - TBARS) but a cause and effect relationship cannot be claimed. Caution is required when interpreting any change in lipid based oxidative stress and its interplay with increases in a protein (mHSP72) whose predominate role is to respond to stress induced changes in protein conformation.

The fifth experimental chapter employed ten consecutive days of once daily hypoxia (75 min, 2980 m) in an attempt to increase basal levels of mHSP72 in excess of that shown in the previous chapter, which utilised a single acute hypoxic exposure. Furthermore, a measures of oxidative stress (plasma TBARS) and EPO secretion were obtained at various time points within the experimental design. Additionally, in line with recent conflicting research investigating the use of intermittent hypoxic training at rest, any changes in maximal oxygen consumption post hypoxic acclimation period were also investigated. Eight healthy recreationally active (mean ± SD: 5.3±1.8 h.wk-1) male subjects (20.2±4.4 yr, 172.1±13.9 cm, 71.1±8.0 kg) volunteered to participate in the study. The hypoxic exposure (75 min, 2980 m) was administered daily for 10 consecutive days, with the exposure commencing and ceasing at 0930 and 1045 respectively. Blood samples were taken immediately pre and post hypoxic exposures on days 1, 2, 3, 4, 5 and 10 for analysis of mHSP72 and plasma TBARS, with EPO specific blood samples taken pre and post hypoxia on days 1, 2, 3, and 10. The maximal oxygen consumption tests were conducted (at sea level) 8 days before and 48 h after the 10 day exposure period. Hypoxic exposure in this manner was sufficient to induce significant (F = 73.2, p < 0.001, partial η2 = 0.92) day-on-day increases in mHSP72 which was proportional to the basal content of the prior day. This increase had a distinct fast (days 1 – 5, 30% increase) and slow (days 5 – 10, 16% increase) phase in accumulation which was seen in tandem with significant (main effects for day (F = 9.0, p = 0.024, partial η2 = 0.60) and time (157.4, p < 0.001, partial η2 = 0.96)) daily transient increases in oxidative stress (plasma TBARS). Within each day, mHSP72 expression was consistently higher after hypoxic exposure (F = 6.2, p = 0.047, partial η2 = 0.51). The plasma TBARS concentration increased significantly (p < 0.05) by around 20% in response to each hypoxic exposure. A significant 5th order polynomial trend was observed for EPO concentration over the 10 days (F = 34.5, p = 0.001, partial η2 = 0.85), characterised by a dramatic 39% increase in EPO concentration the day after the first hypoxic exposure (p = 0.001), followed by a relative plateau over the rest of the hypoxic exposure period, and then a dramatic reduction immediately post-intervention. There were no significant changes observed over the 10 day hypoxic exposure intervention period for absolute maximal oxygen consumption (t = 2.3, p = 0.065) or max heart rate (t = 1.6, p = 0.16), however, there was a small significant increase in time to exhaustion for the incremental test (t = 3.9, p = 0.008, ώ2 = 0.50), though this is likely physiologically negligible. The results within this experimental chapter demonstrated the ability of once daily hypoxia at rest to elicit increases in mHSP72, without the reliance on thermal or mechanical stressors, relied upon previously within the literature to evoke such increases, both in vivo and in vitro. This increase in mHSP72 may be linked to the significant daily increases in oxidative stress (lipid peroxidation – plasma TBARS) but a cause and effect relationship cannot be claimed. Caution is required when interpreting any change in lipid based oxidative stress and its interplay with increases in a protein (mHSP72) whose predominate role is to respond to stress induced changes in protein conformation. The efficacy of increasing basal mHSP72 expression to confer in vivo cellular tolerance to further stressors (exercise, hypoxia, hyperbaria, etc) has not been directly investigated within this chapter and requires further, possibly ex vivo and/or in vivo research.

The sixth and final experimental chapter, utilised five days of once daily hypoxic exposures (the fast phase of accumulation highlighted in the previous chapter) to increase basal mHSP32 and mHSP72. Eight healthy recreationally active (mean ± SD: 6.8±1.8 h.wk-1) male subjects (20.8±3.2 yr, 1.77±15.7 cm, 72.1±11.0 kg, power output at lactate threshold 184±37 W) volunteered to participate in the study. Initial lactate threshold testing on an SRM cycle ergometer allowed lactated threshold to be calculated using the Dmax method with subjects then completing 30 mins of cycling (familiarisation) at 90% of their power output at LT. The hypoxic exposure (75 min, 2980 m) was administered daily for five consecutive days at rest, with the exposure commencing and ceasing at 0930 and 1045 respectively. Blood samples were taken immediately prior to the first hypoxic exposure (hypoxic day 1) and 30 min post final hypoxic exposure (hypoxic day 5). Seven days prior to the hypoxic acclimation period, subjects performed 60 min cycling on a SRM cycle ergometer at 90% of their power output at LT (exercise bout 1 – EXB1), this exercise bout was repeated 1 day post cessation of the hypoxic period (exercise bout 2 – EXB2). Physiological measures of exercise performance (heart rate, ratings of perceived exertion, blood lactate) in EXB1 and EXB2 were recorded throughout to ensure any changes in the biochemical measures of stress protein expression (mHSP72 and mHSP32) and alterations in redox balance (plasma TBARS and glutathione) were not associated with variations in performance intensity between bouts. Blood samples were taken immediately pre and post exercise and 1, 4 and 8 h post exercise for mHSP72 and immediately pre, post and 1 h post exercise for mHSP32, plasma TBARS and glutathione. The five day hypoxic acclimation period was sufficient to reduce the disturbance to redox balance of 60 min prolonged aerobic exercise at 90% of LT. This reduction was evident by the significant increase (32.5%; 95% CI = 19.0% to 45.9%; p < 0.001) in GSSG post EXB1 being absent post EXB2 (p = 0.26). Such a reduction in disturbance to redox balance post exercise is likely attributable to the prior induction (increased content pre EXB2 compared to pre EXB1) and thus bio availability of the potently antioxidant stress protein mHSP32 (p = 0.024) and the highly stress inducible mHSP72 (p < 0.001), in addition to favourable alterations in glutathione ratios. The GSSG was 16.5% lower pre-exercise (95% CI = 3.5% to 29.5%; p = 0.018) and 39.9% lower immediately post-exercise (95% CI = 27.2% to 52.6%; p < 0.001) in EXB2 compared to EXB1. Furthermore, a significant 32.5% increase in GSSG was observed from pre-exercise to immediately post-exercise for EXB1 (95% CI = 19.0% to 45.9%; p < 0.001), whereas no significant change was observed in response to EXB2 (p = 0.26). The hypoxic acclimation period induced significant increases of 2.6 malondialdehyde equivalents in plasma TBARS (95% CI = 1.5 to 3.6 malondialdehyde equivalents; t = 5.6, p = 0.001). Plasma TBARS increased significantly from pre- to post-exercise (p = 0.001) in both EXB1 and EXB2. EXB1 demonstrated an increase of approximately 100% (95% CI = 54.2% to 156.9%; p < 0.001) in mHSP72 immediately post exercise, with values remaining elevated by approximately 50% 1, 4, and 8 h post exercise. The expression kinetics in EXB2, compared to EXB1, did not demonstrate a significant increase (p ≥ 0.79) in mHSP72 immediately post exercise. A similar significant (p = 0.003) EXB1 mediated increase was also observed for mHSP32 immediately post exercise, with the response absent (p ≥ 0.99) immediately post EXB2. The hypoxia mediated increases (approximately 60% (95% CI = 52.9% to 81.4%; t = 11.1, p < 0.001)) in basal mHSP72 remained elevated (approximately 50% higher compared to control and pre EXB1) before the commencement of EXB2. mHSP32 exhibited a similar hypoxia mediated response with increases of approximately 26% (95% CI = 3.2% to 47.6%; t = 2.7, p = 0.03) post hypoxic acclimation period, these elevations where enduring until the commencement of EXB2. The combination of increased bio available mHSP32 and mHSP72 prior to exercise commencing in EXB2 compared to EXB1 may acquiesce the disturbance to redox balance during the second, physiologically identical exercise bout. Furthermore, the favourable alterations in whole blood glutathione redox balance, before commencement of EXB2 compared to EXB1 (i.e. a reduction GSSG and increase in GSH), may, in tandem with elevated basal stress protein levels (mHSP32 and mHSP72), or independently, potentially augment the body‟s ability to deal with exercise induced disturbances to redox balance.

The novel findings within this thesis include the establishment of the quadratic trend in basal mHSP72 expression, which has been shown to be consistently repeatable. This diurnal variation was moderately correlated to core temperature. These findings illustrate the importance of controlling the time of day both interventions and blood samples are administered and collected respectively, specifically, due to basal values being indicatively linked to the magnitude of post stressor mHSP72 expression. Furthermore, the successful use of hypoxia to induce in vivo elevations in basal mHSP72 can be considered novel, as can the demonstration of day on day increases in basal mHSP72 in response to once daily hypoxia (for ten consecutive days). This increase in mHSP72 is the first demonstrated in vivo increase in basal mHSP72 without the reliance on a thermal and/or mechanical stressor. However, objective evidence that such increases in basal mHSP72 have conveyed any tolerance to further stressors has not been obtained. Some support in the final experimental chapter does indicate that a protective adaptation may have occurred, although, the efficacy of increasing basal mHSP72 expression to confer in vivo cellular tolerance to further stressors (exercise, hypoxia, hyperbaria, etc) has not been thoroughly investigated within this thesis and requires further, possibly ex vivo and/or in vivo research. Additionally, there appears to be some evidence that oxidative stress may be a stimulus for increases in mHSP72, as significant increases in TBARS are seen in conjunction with significant elevations in mHSP72 in experimental chapters 4, 5 and 6. This increase in mHSP72 may be linked to the significant daily increases in oxidative stress (lipid peroxidation – plasma TBARS) but a cause and effect relationship cannot be claimed. Caution is required when interpreting any change in lipid based oxidative stress and its interplay with increases in a protein (mHSP72) whose predominate role is to respond to stress induced changes in protein conformation. In further support of a change in redox balance providing a stimulus for increased mHSP72 expression, whole blood glutathione ratios indicate a shift towards a pro-oxidant state have occurred in tandem with increases in mHSP72 and mHSP32 within the final experimental chapter. The absence of such a shift in redox balance (post hypoxic acclimation period) coincided with an absence of significance increases in mHSP72 and mHSP32 expression post exercise. Again, although a cause and effect relationship cannot be claimed to have been established this does provide some evidence that oxidative stress may be a stimulus for hypoxia and exercise induced increases in HSP72. Additionally experimental limitations and lack of prior relevant HSP72 data rendered the use of power/sample calculations not feasible; this is a limitation of the presented thesis. However, the data presented can be used to inform such calculations for future experimental designs. Furthermore, the thesis lacks the use of sham/control groups for experimental chapters 3, 4, 5, and 6 due to economic and logistical restrictions, such groups should be included in future experimental designs. Some chapters may have benefited from use of condition randomisation, but logistical and economic restrictions rendered this unfeasible. Future research of a similar manner is recommended to include such randomisation if possible.

The novel findings presented in this thesis may have a part to play in hypoxia mediated cross tolerance in vivo. Specifically, the potential of the hypoxic protocol utilised to convey tolerance to the oxidative stress associated with sub-maximal aerobic exercise. Such cross tolerance has been postulated within this thesis to originate from hypoxia mediated increased bio available mHSP32 and mHSP72, and favourable glutathione changes within the blood. However, future research should investigate such changes within the blood in tandem to those within the muscle, and expand upon the limited subject numbers used within the experimental chapters of this thesis (data presented can be used for future power/sample calculations). Additionally, attention must be paid to the expanding volume of literature which highlights oxidative stress as a key signalling molecule in the procurement of physiological adaptation (within skeletal muscle and blood). Therefore, it would be likely that a protocol such as the one developed here would be of greater benefit during a period of repeat athletic performance, i.e. within a competitive sporting season, whereby, a reduction in the disturbance to redox balance experienced by an athlete can minimise the recovery time required to return to optimal performance.

Thesis Type	Thesis
Deposit Date	Nov 6, 2015
Publicly Available Date	Feb 23, 2023
Keywords	Sports sciences
Public URL	https://hull-repository.worktribe.com/output/4217280
Additional Information	Department of Sport, Health & Exercise Science, The University of Hull
Award Date	Sep 1, 2010

Monocyte heat shock protein 72 at rest and in response to environmental and exercise stress : implications for cross tolerance in vivo

Taylor, Lee

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