Pilot Study: Comparative Hsp72 Expression in Blood Plasma, Saliva, and Exosomal Fractions During Acute Heat Stress and Acclimation
Authors: Snell Ashanthi , Thornton Staci N. , Szymanski Michael R. , Struder Jeb F. , Filep Erica M. , Morrisey-Basler Margaret , Sekiguchi Yasuki 
Published: May 2024

Objective: To support long-term study biomonitoring during stress, we sought to establish optimized protocols for blood and saliva. Our hypotheses were that 1) stress-specific marker Hsp72 is expressed to suffcient detectable limits and responsive to rest, exercise-heat stress, and to multi-day acclimation in saliva and blood plasma and 2) Hsp72 patterns are distinct in exosome fractions vs. unprocessed whole samples. Methods: Participants (3M/3F, M: 28±4yo, VO2max 48.87±4.08mL·kg−1·min−1, F: 25±2yo, VO2max 41.17±0.74mL·kg−1·min−1) completed a 5-day heat acclimation protocol (~40°C and relative humidity ~40%, ~70% vVO2max jog to reach and maintain a rectal temperature (Trec) range of 38.5-39.5°C for 60 total min). EDTA-treated plasma was separated and a portion was used to fractionate and isolate plasma exosomes for analyses. Whole saliva was processed and fractionated to isolate saliva exosomes for analyses. Hsp72 was analyzed in samples using a high-sensitivity ELISA kit (Enzo Life Sciences). Physiological variables (heart rate (HR), Trec) were used to confirm the stress of the exercise-heat exposures. ANOVA with t-tests for comparisons were used to assess statistical significance (p<0.05). Results: All subjects acclimated, represented by decreased HR (PREHA:107±11.14bpm (M), 170.33±19.50bpm (W), p=0.01) vs. POSTHA:103.67±7.57bpm (M), 140±18.68bpm (W), p=0.04) and decreased Trec post-exercise (PREHA:38.16±0.61°C (M), 39.33±0.50°C (W), p=0.06) vs. POSTHA:37.91±0.41°C (M), 38.39±0.11°C (W), p=0.12). Men and women did differ significantly (p<0.05) with HR changes but were not significantly different (p>0.05) in regards to Trec. Despite physiological acclimation, there were no differences in Hsp72 across time points and by sex. Hsp72 expression was detected in plasma exosomes (0.82-5.18ng·mL−1 (M), 0.48-2.22ng·mL−1 (W)) and saliva exosomes (0.45-10.09ng·mL−1 (M), 0.25-7.18ng·mL−1(W)) and there were no significant differences between men and women (p>0.05) during acute exposures and acclimation, despite some differences in physiological responses. Hsp72 was significantly higher in males overall as compared to females only in saliva exosomes (3.94±3.14ng·mL−1 (M), 1.5±1.27 ng·mL−1 (F), p=0.0003). Conclusion: Subjects acclimatized physiologically without exhibiting Hsp72 changes in their blood plasma or plasma exosomes. Salivary exosomal Hsp72 may be different between men and women. Ongoing research includes validation of the exosome fraction using exosome specific protein markers in co-expression (exosome marker and Hsp72) experiments and ongoing assays of salivary biomarkers of heat stress. Funding: DoD BA200299. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.