# Acute effects of heat intervention and hybrid exercise on protein synthesis, ribosome biogenesis and autophagy

Tom Normand-Gravier, Robert Solsona, Flavie Arnould, Roméo Deriaz, Christelle Bertrand-Gaday, Fabio Borrani, Henri Bernardi, Anthony M.J. Sanchez

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## ► To cite this version:

Tom Normand-Gravier, Robert Solsona, Flavie Arnould, Roméo Deriaz, Christelle Bertrand-Gaday, et al.. Acute effects of heat intervention and hybrid exercise on protein synthesis, ribosome biogenesis and autophagy. Journal of Thermal Biology, 2025, 131, pp.104169. 10.1016/j.jtherbio.2025.104169. hal-05164658

HAL Id: hal-05164658

https://hal.inrae.fr/hal-05164658v1

Submitted on 16 Jul 2025

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# Acute effects of heat intervention and hybrid exercise on protein synthesis, ribosome biogenesis and autophagy

Tom Normand-Gravier<sup>a,b, ID</sup>, Robert Solsona<sup>a ID</sup>, Flavie Arnould<sup>a,b</sup>, Roméo Deriaz<sup>a,b ID</sup>, Christelle Bertrand-Gaday<sup>b ID</sup>, Fabio Borrani<sup>c ID</sup>, Henri Bernardi<sup>b,1 ID</sup>, Anthony M.J. Sanchez<sup>a,c,1,* ID</sup>

## ARTICLE INFO

### Keywords:

- Heat exposure

- Exercise

- Ribosome biogenesis

- Autophagy

## ABSTRACT

The use of kaumatherapy (i.e. heat exposure like sauna-bathing) as passive intervention has become of growing interest to promote skeletal muscle adaptations such as strength gains and preservation of muscle mass. Importantly, the effects of a single exposure to heat (HE) in combination with hybrid exercises, designed to induce both aerobic and resistance adaptations (EX), on protein turnover remains unclear. The objective of this investigation was to evaluate the responses to HE, EX and their combination (EX + HE) on the expression of mRNA and protein related to proteosynthesis, ribosome biogenesis and content, as well as autophagy and cellular stress pathways. Eight-week-old male mice C57BL/6 J mice (n = 8 per condition) underwent acute HE (45min. 40 °C), EX (high-intensity inclined treadmill running) or EX + HE immediately after exercise. Mice were euthanized 240 min post-interventions and quadriceps muscles were harvested for analysis. Acute HE increased the mRNA expression of markers of ribosome biogenesis (POL1RA, UBF), but did not enhance ribosomal content (rRNA 18 S and 28 S) nor ribosomal transcription (pre-rRNA 45 S). Concerning the modulation of protein synthesis, EX induced an increase of MTORC1 downstream targets (P-S6K1, P-RPS6) and protein synthesis flux (assessed by puromycin incorporation) while HE alone did not and post-exercise HE had no additional effects. EX + HE also led to enhanced protein synthesis rates, but did not confer any additional benefit compared to EX alone. Finally, only EX + HE increased the autophagy flux marker LC3-B II/I, the microautophagy marker LAMP2A and phosphorylation level of P-NFκB Ser536. However, no changes in protein carbonylation were detected at this time point. These results suggest that acute post-exercise HE has no additional effects on protein synthesis at 4 h post-exercise but, when combined with EX, increases autophagy and P-NFκB Ser536, suggesting a heightened cellular stress response.

## 1. Introduction

Skeletal muscle hypertrophy and strength gains are common physiological adaptations observed after regular resistance training (RT) (Lopez et al., 2021). These hypertrophic adaptations notably result from activation of the MTORC1 (mechanistic or mammalian target of the

rapamycin complex 1) pathway in myocytes (Philp et al., 2011; McGlory et al., 2017; Solsona et al., 2021). MTORC1 activation promotes hypertrophy by increasing proteosynthesis flux, recruitment of satellite cells, and ribosome biogenesis (de novo synthesis of ribosomes) (Wackerhage et al., 2019; Jin et al., 2019). Acute resistance exercise (RE) modulates the protein response of the main downstream targets of

<footnote><sup>a</sup> University of Perpignan Via Domitia (UPVD), Faculty of Sports Sciences, Laboratoire Interdisciplinaire Performance Santé Environnement de Montagne (LIPSEM), UR4640, 66120, France</footnote>

<footnote><sup>b</sup> DMEM, University of Montpellier, INRAE, Montpellier, France</footnote>

<footnote><sup>c</sup> Institute of Sport Sciences, University of Lausanne, Lausanne, Switzerland</footnote>

<footnote>* Corresponding author. University of Perpignan Via Domitia (UPVD), Faculty of Sports Sciences, Laboratoire Interdisciplinaire Performance Santé Environnement de Montagne (LIPSEM), UR4640, 66120, France.</footnote>

<footnote>E-mail addresses: tom.normand-gravier@univ-perp.fr (T. Normand-Gravier), robert.solsona@univ-perp.fr (R. Solsona), flavie.arnould@etudiant.univ-perp.fr (F. Arnould), romeo.deriaz@gmail.com (R. Deriaz), christelle.bertrand-gaday@inrae.fr (C. Bertrand-Gaday), fabio.borrani@unil.ch (F. Borrani), henri.bernardi@inrae.fr (H. Bernardi), anthony.sanchez@unil.ch (A.M.J. Sanchez).</footnote>

<footnote n="1">¹ co-senior authors.</footnote>
MTORC1 (Bolster et al., 2003). Specifically, phosphorylation of 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) and RPS6 (ribosomal protein S6) increases following a RE bout (Bolster et al., 2003), promoting increased mRNA translation and subsequent hypertrophy with repeated RE stimuli (i.e. training, RT) (Solsona et al., 2021). The hypertrophic response is critically dependent of the MTORC1 activation and phosphorylation of its downstream ribosomal targets (Ogasawara and Suginohara, 2018). Moreover, changes in global protein turnover can explain skeletal muscle mass variations (Goodman et al., 2011). Hence, analyzing protein and organelle turnover is crucial to analyze changes in muscle mass.

Translational efficiency (i.e., protein synthesized per unit of mRNA) and translation capacity (ribosome abundance) also increase with RT (McGlory et al., 2017). Ribosomes are ribonucleoprotein complexes responsible for translating messenger RNA (mRNA) into proteins (Chaillou, 2019). Hence, an increase in ribosomal RNA (rRNA) levels enhances the translational capacity of muscle cells, leading to greater hypertrophy (Solsona and Sanchez, 2020). Total rRNA content positively correlates with the hypertrophic response following RT (Stec et al., 2016). Following active recovery, acute RE increases the expression of ribosomal DNA (rDNA), transcription factors, such as P-TIF (phosphorylated transcription initiation factor-1A), P-UBF (phosphorylated upstream binding factor) and c-Myc (Figueiredo et al., 2016). This suggests that ribosome biogenesis plays a crucial function in promoting muscle hypertrophy (Figueiredo et al., 2015; Wen et al., 2016; Figuereido and McCarthy, 2019) and can be stimulated by RT (Figueiredo et al., 2015).

Conversely, autophagy removes damaged and non-functional cellular components, such as mitochondria and ribosomes, thus preserving muscle health and homeostasis (Sanchez et al., 2014, 2015, 2018; Sanchez, 2016, 2018). Endurance exercise is known to increase autophagic flux, which is critical for promoting adequate cell component turnover (Sanchez et al., 2014). While endurance exercise enhances autophagic flux, which is critical for promoting adequate cell component turnover, the effects of RE on markers on autophagy flux (i.e. LC3-B II/I ratio and P62 content), are less consistent. Interestingly, these markers remain unchanged during RE in rodents (Ato et al., 2017), whereas studies in humans found a decrease of the LC3-B II/I ratio (Fry et al., 2013; Mazo et al., 2021). In addition, MTORC1 is known to inhibit autophagic activity through Ulk1 (Unc-51-like kinase) phosphorylation (Kim et al., 2011). After a single bout of RE, Ulk1 phosphorylation increased, suggesting a downregulation of the autophagic machinery (Ogasawara et al., 2016; Ato et al., 2017). Thus, to date, literature suggests that autophagy may remain unchanged or even be inhibited after a single dose of RE.

Kaumatherapy corresponds to the treatment by heat (Méline et al., 2017; Normand-Gravier et al., 2025) (HE, for heat exposure), such as passive hot-water immersion or sauna bathing, and has been recently studied for its ability to promote muscle adaptations such as hypertrophy and gains in isometric strength (Goto et al., 2011; Méline et al., 2021). Repeated passive HE may be a successful approach for increasing muscle strength, the effects being sometimes comparable to exercise in sedentary populations (Rodrigues et al., 2020). Moreover, the beneficial impact of HE has been recently highlighted during exercise training programs and during skeletal muscle atrophy (Normand-Gravier et al., 2025). Accordingly, acute passive muscle heating (i.e., heat applied without muscle contraction) can increase the phosphorylation levels of Akt, MTOR and RPS6 (Kakigi et al., 2011; Ihsan et al., 2020) and increases mitochondrial enzyme content and activity in humans (Marchant et al., 2023). Thus, by mimicking several cellular processes involved in responses to exercise, HE may help to prevent skeletal muscle atrophy in sedentary individuals or those affected by disease, by stimulating protein synthesis and supporting mitochondrial homeostasis (Rodrigues et al., 2020; Normand-Gravier et al., 2025).

Limited research exists on the acute effects of HE following exercise on cellular pathways associated with protein synthesis, autophagy and

ribosome biogenesis. HE after resistance exercise can enhance phosphorylation levels of protein synthesis markers, such as Akt (protein kinase B), MTOR (mammalian/mechanistic target of rapamycin), 4E-BP1 and S6K1 (S6 kinase 1) (Kakigi et al., 2011). Regarding endurance exercise, it has been reported increased phosphorylation of S6K1 and p38 MAPK (p38 mitogen-activated protein kinase) when mice were exposed to an environmental chamber set to 40 °C for 30 min immediately following a 30-min treadmill running session (Tamura et al., 2014). However, no study has evaluated the effects of a single exposure to HE on ribosome biogenesis and autophagic markers nor its application after an exercise protocol allowing the development of both muscle growth and endurance.

Therefore, this study aimed to investigate acute responses to a hybrid exercise (EX) that we called "Growth Power Training" (GPT), acute HE and their sequential application (EX + HE) on protein synthesis, ribosome biogenesis and autophagy markers. We hypothesized that (i) HE would increase the expression of protein turnover markers similarly to EX and (ii) the sequential application of EX + HE would have an additive effect on these pathways.

## 2. Methods

### Ethical approval

The experiments were performed in accordance with European directives and approved by (i) the Ethical Committee of Region Languedoc-Roussillon (number C2EA-36) and (ii) the French Ministry of Higher Education and Research (#43054_2,023,042,015,168,782).

### 2.1. Animals and procedures

32 eight-week-old C57BL/6 J male mice (Janvier Labs, Saint-Berthevin, France) have been used for this study. Animals were housed individually in a controlled environment room (22 °C ± 1 °C) with a 12:12-h LD cycle (dark period from 10 a.m. to 10 p.m.). All animals had a standardized diet (A04, SAFE, Augy, France) and water ad libitum. Following a 2-week adjustment period to their new circadian rhythm and environment, mice performed one week of running habituation on EXER-3/6 Treadmill, Linton Instrument (3 sessions of 10 min, 5–10 m/min, slope 0–25°). Then, animals have been assigned to one of the four groups: CTL (control, N = 8), EX (exercise, N = 8), HE (heat exposure, N = 8), EX + HE (exercise + heat exposure, N = 8). The details of exercise and heat interventions are provided below, and the study design is detailed in Fig. 1. Four hours after the experiment, animals were euthanized, without anaesthesia, by cervical dislocation. Quadriceps muscles were collected, weighed, and frozen in liquid nitrogen. Samples were stored at –80 °C before analysis. For rectal temperature measurement, mice were hand-restrained and placed on a cage lid. The tail was gently lifted and a rectal probe thermometer (RET-3 Physitemp) lubricated with vaseline was carefully inserted 20 mm into the rectum from the base of the tail. Positioning the mouse for rectal probe insertion took approximately 15 s. After insertion, the probe was left in place for 5 s, allowing temperature stabilization.

### 2.2. Growth Power Training (GPT)

One week before the Growth Power Training exercise (EX) and one week after treadmill running habituation, mice were subjected to a supra-maximal speed (SMS) test to adjust and individualize the training load on a motor-driven treadmill (Seldeen et al., 2018). After a 5-min warm-up (8 m/min), the mice were subjected to run intervals of 20 s, interspersed with a 40-s period of relative rest (5 m/min). This incremental test started at a velocity of 11 m/min and was conducted until exhaustion (i.e. the time where mice were unable to run continuously and touched the experimenter's hand (placed at the end of the treadmill) three times in succession). SMS was recorded as the last interval that was
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Schematic overview of the protocol. After one week of treadmill habituation (HAB), mice were subjected to a supra-maximal speed test (SMS). One week after the SMS test, mice realized either a high-intensity inclined treadmill running session (EX), heat exposure (HE) or EX + HE. Quadriceps muscles were dissected 4 h after the completion of EX, HE or EX + HE.</figcaption></figure>

successfully completed before failure. One week after the SMS test, the mice completed a high-intensity inclined treadmill running (EX) session. After a 5-min initial warm-up (8 m/min) and progressive acceleration until SMS, mice were subjected to 15 intervals of 20 s at SMS interspersed with a 40-s period of active recovery (5 m/min). For SMS and EX sessions, the treadmill was inclined at 25°. This inclined treadmill running training protocol has been used previously to promote long-term muscle hypertrophic gains (Seldeen et al., 2018; Goh et al., 2019) and was qualified as an adequate method to promote these adaptations (Murach et al., 2020). However, due to the multiple intervals, EX can also induce endurance-related adaptations and can be considered as a hybrid protocol that promotes both endurance and resistance adaptations such as the protocol conceived by Murach et al., (2020). SMS was not significantly different between EX and EX + HE (29.25 m/min ±3.94 and 27 m/min ±4.94, respectively).

## 2.3. Heat intervention

Mice were placed with their cages in a hot environmental chamber. The heat exposure lasted for 45 min, and the temperature was set to 40 °C. They always had access to water and food during HE. HE was conducted immediately after EX in the EX + HE group. CON and EX animals were exposed to a normal climate-controlled room (22 °C). This heat intervention was adapted from a previous study, showing a significant rise in rectal temperature (≈39–40 °C) and effects on S6K1 phosphorylation and mitochondrial enzyme activity (Tamura et al., 2014).

## 2.4. Assessment of protein synthesis

The Surface Sensing of Translation (SUnSET) method was used to evaluate protein synthesis in vivo (Schmidt et al., 2009). This method involves the use of anti-Puromycin antibody, and has been validated to accurately measure changes in total proteosynthesis (Goodman and Hornberger, 2013). Puromycin was injected intraperitoneally with puromycin exactly 20 min before death.

## 2.5. qRT-PCR analysis

50 mg of tissue was collected for RNA extraction. TRIzol Reagent was used according to the manufacturer's instructions. RNA quality (260/280 ≥ 1.8) and concentration were assessed for each sample by spectrophotometry (BioDrop, Fisher Scientific). Total RNA (1 µg) was reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The primer sequences used for gene expression (Table 1) were designed using Primer-BLAST (NCBI). Agarose electrophoresis and melting curve analyses were performed for each primer to assess primer specificity. Primer efficiency was assessed by using serial dilutions to calculate the slope of the standard curve. This ranged from 80 % to 108 %. qRT-PCR (quantitative reverse polymerase chain reaction) was carried out with SensiFAST SYBR Hi-ROX Mix (Bioline, USA), on a thermocycler (StepOne real-time PCR system, Applied Biosystems, USA): 2 min at 95 °C, 40 cycles of 5 s for denaturation at 95 °C, and 15 s at 60 °C for annealing and extension. Samples were run on clear-well plates for target genes, with a human genomic DNA contamination control and a reverse transcription on each plate. Gene expression was normalized using the housekeeping gene β2-microglobulin (β2MG) that is known to be very stable in adult muscles of mice (Ma et al., 2022). Calculation of gene expression (i.e. mRNA content) was carried out with the comparative cycle threshold method (2⁻ΔΔCt) using Applied Biosystems StepOne Software (version 2.3).

## 2.6. Western Blot analysis

30 mg of tissue was collected for protein extraction. Powdered muscles were homogenized in lysis buffer [1 % Triton X-100; 20 mM MOPS, pH 7; 5 mM EDTA; 2 mM EGTA; 30 mM NaF; 20 mM sodium pyrophosphate; 60 mM β-glycerophosphate; 1 mM sodium orthovanadate; 1 mM dithiothreitol; and a cocktail of protease inhibitors (Roche, Ref 11,836,153,001)]. Homogenates were sonicated on ice 4 × 10 s to shear nuclear DNA. Then, samples have been centrifuged at 15,000 g (10 min, 4 °C). Supernatant was collected and protein concentration was determined by Bradford method (Bio-Rad, Ref 5,000,001). Thereafter, 50 µg of protein was diluted in Laemmli sample buffer, resolved under denaturing condition by SDS-PAGE, and blotted onto nitrocellulose support matrix (Amersham, Protran Premium, 0.2 µm). Membranes were stained with Ponceau S and then blocked in 5 % non-fat dry milk in TBST (0.1 % tween) wash buffer during 2 h. Then, membranes were

<table><caption>**Table 1**

List of primers used for q-PCR.</caption><thead><tr><th>Gene</th><th>Forward primer (5' to 3')</th><th>Reverse primer (5' to 3')</th></tr></thead><tbody><tr><td>Pre-rRNA<br>45 S</td><td>CTCTTAGATCGATGTGGTGCTC</td><td>GCCCGCTGGCAGAACGAGAAG</td></tr><tr><td>rRNA 18 S</td><td>AAACGGCTACCACATCCAAG</td><td>GCTGGAATTACCGCGGCT</td></tr><tr><td>rRNA 28 S</td><td>GCGGGTGGTAAACTCCATCT</td><td>CACGCCCTCTTGAACTCTCT</td></tr><tr><td>ATG2</td><td>CTGTTGCCCAACATCCACCTGA</td><td>CAAGTGTCTCCACTGGTGAAGG</td></tr><tr><td>B2MG</td><td>TTCTGGTGCTTGTCTCACTGA</td><td>CAGTATGTTCGGCTTCCCATTC</td></tr><tr><td>GABARAP</td><td>GTGGAGAAGGCTCCTAAAGCCA</td><td>AGGTCTCAGTGGATCCTCTTC</td></tr><tr><td>HSP70</td><td>GAAGGTGCTGGACAAGTGC</td><td>GCCAGCAGAGGCCTCTAATC</td></tr><tr><td>HSP90</td><td>TGGGTTACATGGCAGCAAAG</td><td>TGTTAGCATGGGTCTGGGGA</td></tr><tr><td>NRF2</td><td>CCGCTACACCGACTACGATT</td><td>ACCTTCATCACCAACCCAAG</td></tr><tr><td>P62</td><td>GCCAGAGGAACAGATGGAGT</td><td>TCCGATTCTGGCATCTGTAG</td></tr><tr><td>PARKIN</td><td>TCAGAAGCAGCCAGAGGTC</td><td>TCTGAGGTTGGGTGTGCTC</td></tr><tr><td>PGC1-α</td><td>GGAGCCGTGACCACTGACA</td><td>TGGTTTGCTGCATGGTTCTG</td></tr><tr><td>PINK1</td><td>CACACTGTTCCTCGTTATGAAGA</td><td>CTTGAGATCCCGATGGGCAAT</td></tr><tr><td>POL1RA</td><td>GCTGACTGGAACTTCTCTCGT</td><td>CGCAGGTAGCGCATACTTCT</td></tr><tr><td>TIF-1A</td><td>ATTTTGAGCGCATTGTGTTGAGC</td><td>GGGAGCATCTGGCGACTGTTC</td></tr><tr><td>UBF</td><td>GTTCAGGGAGAACCCAAG</td><td>TTGACCCAGAGGTCCAAGTG</td></tr><tr><td>ULK2</td><td>GAGGACGAAGACTCTCTACTGG</td><td>GAGTGCTACTCCTGGCTTCAT</td></tr><tr><td>VEGF</td><td>CACGACAGAAGGAGAGCAGA</td><td>ATCAGCGGCACACAGGAC</td></tr></tbody></table>

B2MG housekeeping gene was used as a reference gene.
incubated overnight at 4 °C in either 5 % BSA or non-fat dry milk in TBST (0.1 % tween) following supplier recommendations and using primary antibodies against Puromycin (dilution 1:2000, #MABE343, Millipore, USA), Ubiquitin (dilution 1:1000, #z0458, Dako, USA), HSP27 (dilution 1:750, #SPA-800, Enzo, USA), HSP70 (dilution 1:750, #SPA-810, Enzo, USA), LAMP2A (dilution 1:500, #ab18528, Abcam, UK), DNPH (dilution 1:150, #S7150, Millipore, USA). Other antibodies were from Cell Signaling (USA): P-S6 Ribosomal Protein (P-RPS6) Ser240/244 (dilution 1:1000, #5364), P-S6K1 Thr389 (dilution 1:1000, #9206), P-MTOR Ser 2448 (dilution 1:1000, #2971), P-4E-BP1 Thr37/46 (dilution 1:1000, #2855), P-AMPK Thr172 (dilution 1:750, #50081), P-DRP1 Ser616 (dilution 1:500, #3455), P-NFκB Ser536 (dilution 1:500, #3033), P-ULK1 Ser757 (dilution 1:500, #14202), ATG7 (dilution 1:500, #8558), LC3-B (dilution 1:500, #2775), SQSTM1/P62 (dilution 1:500, #5114), and P-EIF2α Ser51 (dilution 1:1000, #3398). Normalization of total and phospho-protein was performed using Ponceau S staining (Sander et al., 2019). Detection has been carried out using HRP-linked secondary antibodies (dilution 1:2000, #7074 for rabbit, #7076 for mouse and #7077 for rat, Cell Signaling). Immunoblots were revealed using a Femto West HRP substrate kit (GenTex, Ref GTX14698). Finally, proteins have been visualized by enhanced chemiluminescence. Proteins have been quantified using Image Lab software (Version 5.2.1., Biorad).

## 2.7. Immunoblot analysis of protein carbonyls

Protein carbonylation was assessed by measuring the levels of carbonyl groups using the OxyBlot Protein Oxidation Detection kit (Merck, S7150), according to the manufacturer's instructions. Briefly, 20 µg of solubilized protein were derivatized with 2,4-dinitrophenylhydrazine (DNPH) under acid denaturing conditions. To control for nonspecific antibody binding, separate aliquots of protein were acid-denatured but not treated with DNPH. Denatured proteins were resolved by SDS-PAGE on an 8 % polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were stained with Ponceau S then blocked for 1 h in 1 % BSA in PBST (0.05 % tween). Blots were incubated overnight at 4 °C with the first antibody specific for DNPH supplied in the kit, diluted in 1 % BSA in PBST. After washing, membranes were incubated with the second antibody supplied in the kit for 1 h at room temperature. Immunoblots were revealed using a Femto West HRP substrate kit and quantified using Image Lab software.

## 2.8. Statistical analysis

Jamovi (version 2.3.21) and RStudio (version 2024.12.0) were used for statistical analyses. Data are expressed as boxplots with mean, median, quartiles and individual data represented graphically. The threshold of statistical significance was set at 0.05. When conditions were met (p-value >0.05 for Shapiro-Wilk and Levene tests), one-way ANOVA was used, and Tukey's post hoc multiple comparisons tests were utilized when p-value was <0.05 for the Fisher test. When conditions were not met, a non-parametric test (Kruskal-Wallis) was used and DSCF's post hoc tests were performed when p-values were <0.05 for the Kruskal-Wallis test. When p-values were <0.05, effect size was measured with Cohen's d for one-way ANOVA and Rosenthal's r for Kruskal-Wallis.

# 3. Results

## 3.1. Rectal temperature

Average rectal temperature was 36.5 ± 0.5 °C for CTL, 38.5 ± 0.2 °C for EX, 39.7 ± 0.3 °C for HE and 39.9 ± 1.3 °C for EX + HE. Rectal temperature was higher for HE, EX and EX + HE, compared with CTL (P = 0.009, r = 0.91 for HE; P = 0.009, r = 0.91 for EX and P = 0.009, r = 0.90 for EX + HE). Moreover, rectal temperature was significantly

elevated for HE (P = 0.009, r = 0.91) and EX + HE (P = 0.035, r = 0.78), compared to EX.

## 3.2. Protein synthesis flux and protein turnover markers at the protein level

Global protein synthesis, assessed via puromycin incorporation, increased almost 2-fold for EX and more than 2-fold for EX + HE (P = 0.004, r = 0.90 and P = 0.009, r = 0.84, respectively), but not for HE (P = 0.997), compared to CTL (Fig. 2A). Regarding ubiquitin, no statistical significance was found between the groups (Fig. 2B).

No statistical significance was found between groups for P-MTOR (Fig. 3A). P-S6K1 expression increased more than 4-fold in EX and EX + HE (P = 0.006, r = 0.90 and P = 0.007, r = 0.90, respectively), but not for HE (P = 0.305), compared with CTL (Fig. 3B). No statistical significance was observed between groups for P-4E-BP1 (Fig. 3C) and P-EIF2α (Fig. 3D). The phosphorylation level of RPS6 increased almost 2-fold for EX (P = 0.017, r = 0.79), but did not differ in the HE and EX + HE groups (P = 0.975 and P = 0.655, respectively), compared to CTL (Fig. 3E).

Regarding autophagy, ATG7 increased more than 3-fold in the EX and EX + HE groups (P = 0.004 and r = 0.90 for both groups), compared with CTL. ATG7 was also increased in the EX and EX + HE groups (P = 0.017 and r = 0.79 for both groups), compared with HE alone (Fig. 4A). LC3-B II/I ratio increased almost 2.5-fold in EX + HE (P = 0.006, r = 0.87), but was unchanged for HE and EX (P = 0.975 and P = 0.997, respectively), compared to CTL. LC3-B II/I ratio was also increased for EX + HE compared to EX alone (P = 0.017, r = 0.79) (Fig. 4B). No statistical significance was found between groups for P62 (Fig. 4C). LAMP2A increased more than 2.5-fold in EX and EX + HE (P = 0.007 and r = 0.90 for both groups), compared with CTL. LAMP2A was also increased for EX (P = 0.012, r = 0.81) and EX + HE (P = 0.009, r = 0.84), compared with HE alone (Fig. 4D). Finally, P-ULK1 increased almost 2-fold for EX (P = 0.045, d = 1.44) and EX + HE (P = 0.023, d = 1.59), but not for HE (P = 0.852), compared with CTL. No difference was observed between EX and EX + HE (P = 0.990) (Fig. 4E).

## 3.3. rRNA and mRNA expression of protein turnover markers and ribosome biogenesis

Pre-rRNA 45 S was significantly upregulated for EX (P = 0.003, d = 1.96) and EX + HE (P = 0.005, d = 1.84), compared to CTL. Its expression was also significantly upregulated for EX (P < 0.001, d = 2.76) and EX + HE (P < 0.001, d = 2.63), compared to HE. rRNA 18 S was significantly downregulated for HE (P = 0.006, r = 0.87) and EX + HE (P = 0.004, r = 0.90), compared to CTL. rRNA 28 S was significantly downregulated for HE, EX and EX + HE (P = 0.004, r = 0.90 for all groups), compared to CTL (Table 2).

Autophagic markers ATG2 and ULK2 were significantly upregulated for HE, compared to CTL (P = 0.014, r = 0.90 and P = 0.006, r = 0.90, respectively for each marker). ATG2 and ULK2 were also increased for EX, compared to CTL (P = 0.017, r = 0.80 and P = 0.023, r = 0.76, respectively for each marker). However, in the EX + HE group, none of the autophagic markers were increased, compared with CTL. Moreover, gene expression of ULK2 was downregulated for EX + HE, compared with the HE group (P = 0.007, r = 0.90) (Table 2).

UBF expression, a ribosome biogenesis marker, increased in both the HE and EX groups, compared to CTL (P = 0.006, r = 0.90 and P = 0.049, r = 0.70, respectively). UBF content was also enhanced for HE, compared to EX + HE (P = 0.007, r = 0.90). POL1RA expression followed a similar trend, with higher levels in both the HE and EX groups, compared to CTL (P = 0.004, d = 1.97 and P = 0.012, d = 1.68, respectively). In addition, POL1RA content was also higher for HE and EX, compared with EX + HE (P = 0.004, d = 1.95 and P = 0.013, d = 1.66, respectively) (Table 2).

NRF2 increased in both the HE and EX groups, compared to CTL (P = 0.006, r = 0.90 and P = 0.020, r = 0.79, respectively). The
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Effects of heat exposure and exercise on protein synthesis (puromycin incorporation) and protein ubiquitination (total ubiquitinated proteins profile). Data are presented as means ± SD, *, **, *** significantly different compared to the control group; §, §§, ### significantly different between HE and EX + HE groups; #, ##, ### significantly different between HE and EX group; $, SS, SSS significantly different between EX + HE and EX group (P < 0.05, P < 0.01, P < 0.001, respectively). CTL = Control; HE = Heat Exposure; EX = Exercise.</figcaption></figure>

mitochondrial biogenesis marker PGC1-α increased in all experimental conditions compared to CTL (P = 0.006, r = 0.90; P = 0.007, r = 0.90 and P = 0.004, r = 0.90 for HE, EX and EX + HE, respectively). Notably, PGC1-α content was significantly higher in EX, compared to HE (P = 0.009, r = 0.90). The ubiquitin kinase PINK1 showed increased expression for HE, compared to EX and EX + HE (P = 0.010, r = 0.87 and P = 0.006, r = 0.90). Similarly, PARKIN expression was higher for HE, compared to EX and EX + HE (P = 0.028, r = 0.77 and P = 0.007, r = 0.90, respectively) and to CTL (P = 0.007, r = 0.90). (Table 2).

Finally, no statistical significance was found between groups for GABARAPL1, P62 and VEGF expression (Table 2).

### 3.4. Cellular stress response

At the mRNA level, Heat Shock Protein 70 expression increased in all experimental conditions compared to CTL (P = 0.007, r = 0.90; P = 0.004, r = 0.90 and P = 0.011, r = 0.89 for HE, EX and EX + HE, respectively), but HSP70 content was significantly higher in HE and EX + HE, compared to EX (P = 0.028, r = 0.77 and P = 0.016, r = 0.86, respectively). Similarly, HSP90 content increased in HE and EX + HE (P < 0.001, d = 2.43 and P = 0.004, d = 1.91, respectively), compared to CTL, and with higher expression found in HE, compared to EX (P = 0.011, d = 1.75) (Table 2.).

No statistical significance was found between groups for P-AMPK (Fig. 5A) and P-DRP1 (Fig. 5B) but a tendency toward an increase was observed for P-DRP1 in EX + HE group, compared to CTL (P = 0.066). P-NFκB increased more than 2-fold in EX + HE (P = 0.005, d = 2.02) and 2-fold in EX (P = 0.023, d = 1.67), compared with CTL. P-NFκB was also increased in EX and EX + HE (P = 0.002, d = 2.12 and P < 0.001, d = 2.47, respectively), compared with HE alone (Fig. 5C). HSP27 decreased more than 2-fold in EX (P = 0.032, d = 1.47), but not in HE (P = 0.974) and EX + HE (P = 0.095), compared with CTL (Fig. 5D). No statistical significance was found between groups for HSP70 (Fig. 5E).

Finally, carbonyl content, determined using DNPH detection to

measure overall oxidative stress, showed no significant difference between the groups (Fig. 6).

### 4. Discussion

This study aimed to investigate the acute effects of exercise (EX), heat exposure (HE), and their sequential application (EX + HE) on markers of skeletal muscle protein turnover at both gene and protein levels. Key findings revealed that while EX increased proteosynthesis markers and global protein synthesis, HE alone failed to elicit a comparable response. However, the combination of EX and HE demonstrated the most pronounced upregulation of autophagy flux and a cellular stress marker, as evidenced by the LC3B-II/I ratio and P-NFκB, suggesting that combined stimuli amplified cellular stress at this specific time point. Notably, HE alone increased mRNA expression of ribosomal biogenesis and autophagy-related markers to levels similar to those induced by EX. Conversely, sequential EX + HE application down-regulated mRNA expression in these pathways, highlighting a potential interaction effect between stressors, even if ribosomal transcription (pre-rRNA 45 S) was increased to the same extent as with EX.

#### 4.1. EX and EX + HE increased global protein synthesis

Muscle protein synthesis, driven by ribosome biogenesis or enhanced MTORC1 signaling, is crucial to promote muscle hypertrophy (Roberts et al., 2023). We report here that acute HE alone did not increase total protein synthesis rates whereas EX and EX + HE increased it significantly. These findings align with previous studies demonstrating that acute RE alleviates protein synthesis flux (West et al., 2016, 2019; Langer et al., 2022). Hence, our high-intensity inclined treadmill protocol stimulates anabolic signaling similarly to RE, consistent with evidence that acute high-intensity interval and aerobic exercises increase muscle proteosynthesis (Bagheri et al., 2022). However, post-exercise HE did not lead to an additional effect on global protein synthesis rate
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Effects of heat exposure and exercise on MTOR Pathway (P-MTOR, P-S6K1, P-4E-BP1, P-EIF2α and P-RPS6). Data are presented as means ± SD. *, **, *** significantly different compared to the control group; §, §§, §§§ significantly different between HE and EX + HE groups; #, ##, ### significantly different between HE and EX group; $, $$, $$$ significantly different between EX + HE and EX group (P < 0.05, P < 0.01, P < 0.001, respectively). CTL = Control; HE = Heat Exposure; EX = Exercise.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Effects of heat exposure and exercise on autophagy (ATG7, LC3-B II/I, P62, LAMP2A, P-ULK1). Data are presented as means ± SD. *, **, *** significantly different compared to the control group; §, §§, §§§ significantly different between HE and EX + HE groups; #, ##, ### significantly different between HE and EX group; $, $$, $$$ significantly different between EX + HE and EX group (P < 0.05, P < 0.01, P < 0.001, respectively). CTL = Control; HE = Heat Exposure; EX = Exercise.</figcaption></figure>
<table><caption>**Table 2**
Q-PCR analysis.</caption><thead><tr><th>Gene</th><th>CTL</th><th>HE</th><th>EX + HE</th><th>EX</th></tr></thead><tbody><tr><td><strong>Ribosome</strong></td><td>1.00 ± 0.21</td><td>0.69 ± 0.40</td><td>1.70 ± 0.51 **, §§§</td><td>1.75 ±</td></tr><tr><td><strong>Content</strong></td><td>1.00 ± 0.04</td><td>0.64 ± 0.25</td><td>0.68 ± 0.13 **</td><td>0.34 **, ***</td></tr><tr><td>Pre-rRNA</td><td>1.00 ± 0.05</td><td>**</td><td>0.53 ± 0.23 **</td><td></td></tr><tr><td>45 S</td><td></td><td>0.45 ± 0.21</td><td></td><td>0.72 ±</td></tr><tr><td>rRNA 18 S</td><td></td><td>**</td><td></td><td>0.28</td></tr><tr><td>rRNA 28 S</td><td></td><td></td><td></td><td>0.50 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.25 **</td></tr><tr><td><strong>Autophagy</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>ATG2</td><td>1.00 ± 0.24</td><td>3.32 ± 1.19 *</td><td>1.54 ± 1.26</td><td>2.79 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>1.59 *</td></tr><tr><td>GABARAP</td><td>1.00 ± 0.24</td><td>1.66 ± 0.50</td><td>1.07 ± 0.50</td><td>1.44 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.69</td></tr><tr><td>P62</td><td>1.00 ± 1.46</td><td>1.49 ± 0.50</td><td>0.77 ± 0.40</td><td>1.00 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.47</td></tr><tr><td>ULK2</td><td>1.00 ± 0.19</td><td>2.24 ± 0.26 **</td><td>1.18 ± 0.47 §§</td><td>1.64 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.57 *</td></tr><tr><td><strong>Ribosome biogenesis</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>POL1RA</td><td>1.00 ± 0.28</td><td>2.27 ± 0.66 **</td><td>1.02 ± 0.65 §§</td><td>2.08 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.85 *, §</td></tr><tr><td>TIF-1A</td><td>1.00 ± 0.19</td><td>1.87 ± 0.85</td><td>1.15 ± 0.60</td><td>1.71 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.78</td></tr><tr><td>UBF</td><td>1.00 ± 0.18</td><td>2.10 ± 0.78 **</td><td>1.02 ± 0.35 §§</td><td>1.53 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.47 *</td></tr><tr><td><strong>Mitochondrial function</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>NRF2</td><td>1.00 ± 0.15</td><td>2.34 ± 0.70 **</td><td>1.30 ± 0.54</td><td>1.83 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.64 *</td></tr><tr><td>PARKIN</td><td>1.00 ± 0.22</td><td>2.51 ± 2.02 **</td><td>0.90 ± 0.31 §§</td><td>1.02 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.40 #</td></tr><tr><td>PGC1-α</td><td>1.00 ± 0.14</td><td>2.35 ± 0.76 **</td><td>4.83 ± 2.73 **</td><td>6.78 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>2.46 **, **</td></tr><tr><td>PINK1</td><td>1.00 ± 0.56</td><td>1.59 ± 0.27</td><td>0.65 ± 0.21 §§</td><td>0.88 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.28 #</td></tr><tr><td><strong>Angiogenesis</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>VEGF</td><td>1.00 ± 0.99</td><td>1.59 ± 0.56</td><td>1.50 ± 0.68</td><td>1.86 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>1.10</td></tr><tr><td><strong>Heat Shock Proteins</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>HSP70</td><td>1.00 ± 0.31</td><td>50.65 ± 52.30 **</td><td>58.67 ± 36.94 *</td><td>10.89 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>9.41 **, #, §</td></tr><tr><td>HSP90</td><td>1.00 ± 0.65</td><td>3.34 ± 1.26 ***</td><td>2.84 ± 1.25 **</td><td>1.66 ±</td></tr><tr><td></td><td></td><td></td><td></td><td>0.47 #</td></tr></tbody></table>

B2MG housekeeping gene was used as a control. Data were analysed using the 2⁻ΔΔc~t~ method of fold change relative to the control group. *, **, *** Significantly different compared to the control group (P < 0.05, P < 0.01, P < 0.001, respectively). §, §§, §§§ Significantly different between the HE and EX + HE (P < 0.05, P < 0.01, P < 0.001, respectively). #, **, *** Significantly different between HE and EX (P < 0.05, P < 0.01, P < 0.001, respectively). §, §§, §§§ Significantly different between EX + HE and EX (P < 0.05, P < 0.01, P < 0.001, respectively).

compared to EX alone. This result is in line with another study that found that post-exercise hot-water immersion did not further enhance myofibrillar proteosynthesis, compared to RE alone in healthy young males (Fuchs et al., 2020). Furthermore, no impact of hot-water immersion or post-exercise hot-water immersion was recently observed on muscle protein synthesis rates in rats after RE (Kotani et al., 2024). Therefore, as discussed by Kotani and colleagues (Kotani et al., 2024), the heat-induced hypertrophy (Goto et al., 2011) cannot be explained by an acute activation of proteosynthesis.

Protein breakdown, which is partially modulated by the ubiquitin-proteasome system, degrades ubiquitinated proteins and enhances subsequent muscle atrophy. In the current study, total ubiquitinated protein levels were not decreased following EX and showed a similar trend with EX + HE. This result differs from previous findings (Kotani et al., 2024) where a small decrease in ubiquitinated proteins was found 3 h post-RE. However, no effect of post-exercise hot water immersion

(HWI) was found, which is consistent with our findings, suggesting that HE and post-exercise HE have no additional effects on the total ubiquitinated proteins profile following acute exercise. Thus, HE does not appear to have additive effects on global protein turnover.

The MTORC1 axis, a key pathway for translation of mRNA (Sanchez et al., 2019; Solsona and Sanchez, 2020) is enhanced by RE, with or without passive muscle heating (Kakigi et al., 2011; Fry et al., 2011; Chaillou et al., 2014; Mazo et al., 2021). Furthermore, RPS6 and 4 E-BP1 phosphorylation is strongly correlated with protein synthesis (Stewart and Thomas, 1994; Fry et al., 2011). In this study, acute HE had no effect on P-MTOR, P-RPS6 or P-4E-BP1, which contrasts with previous reports (Kakigi et al., 2011; Yoshihara et al., 2013). This opposite result might be explained by the timing of muscle sampling (i.e. <1 h vs 4 h post-HE). Phosphorylation events occur rapidly (<1 h) after RE or HE (Dreyer et al., 2006; Kakigi et al., 2011; Yoshihara et al., 2013), whereas muscle biopsies were collected later in our protocol. Supporting this hypothesis, Kotani et al. found no increase in P-RPS6 or P-S6K1 3 h post HWI but observed increases in P-RPS6 and P-4E-BP1 at 24 h post HWI (Kotani et al., 2024), suggesting that acute responses to HE follows precise kinetics. In our study, in contrast to HE, only EX significantly increased P-RPS6, suggesting an increase in protein synthesis. Hence, translation kinetics might differ between HE and EX, with high-intensity exercise providing a more potent anabolic stimulus. Additionally, it is suggested that an increase in P-RPS6 modifies ribosome function and leads to a translation rate increase in mRNAs encoding sarcomeric proteins (Chaillou et al., 2014). EIF2α is a translation initiation factor that downregulates protein synthesis when phosphorylated at Ser51 (Kimball, 1999). In our study, P-EIF2α was unchanged following acute HE, EX, and EX + HE, which is consistent with previous reports of no change in P-EIF2α at 1 h post-acute RE (Bolster et al., 2003) or at 3 and 6 h post-acute RE (Drummond et al., 2008). Collectively, and given the lack of significant differences observed in the puromycin incorporation data, our findings indicate that combining our hybrid exercise with HE failed to elicit a synergistic anabolic response compared to EX alone.

## 4.2. Coupling exercise with heat exposure leads to increased autophagy, but contrasting results were found on cellular stress responses

Autophagy acts as a major regulator of skeletal muscle homeostasis by removing damaged cellular components (Sanchez et al., 2012, 2014; Sanchez, 2016). Furthermore, it has been suggested that an adequate autophagy level is of importance for skeletal muscle maintenance in mice (Nair and Klionsky, 2011). Acute endurance exercise rises markers of autophagy activity (Sanchez et al., 2014) whereas RE seems to not have these effects (Fry et al., 2013; Ato et al., 2017; Mazo et al., 2021). In the current study, it is important to note that no autophagic or cellular stress markers increased following HE. Interestingly, only EX + HE raised the ratio LC3-B II/I, a finding that contrasts with other works (Fry et al., 2013; Ato et al., 2017; Mazo et al., 2021), which reported no increase in the expression of this marker following exercise alone. This increase in LC3-B II/I seems to indicate that the combined stress of GPT and HE enhances autophagic flux, likely limiting the accumulation of damaged organelles (i.e. mitochondria, ribosomes). However, two other autophagic markers, ATG7 and LAMP2A, were significantly increased for EX + HE but also in the EX group, suggesting that exercise alone also promotes the removal of damaged organelles by activating macro-autophagy and chaperone-mediated autophagy. Interestingly, although these autophagic markers were elevated in the EX and EX + HE groups, the autophagy initiation phase appears to be reduced, as indicated by increased levels of phosphorylated ULK1 at Ser757 in both experimental conditions. In this sense, the timepoint of our study (i.e. 4 h post-exercise) might be an inflection point between ULK1 and MTOR activation in EX and EX + HE conditions. NFκB is considered as a master regulator of inflammation by activating pro-inflammatory cytokines and chemokines in response to various stressors including exercise and environmental challenge (Vella et al., 2012; Liu et al., 2017). Hence, an
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Effects of heat exposure and exercise on cellular stress responses (P-AMPK, P-DRP1, P-NFkB, HSP27 and HSP70). Data are presented as means ± SD. *, **, *** significantly different compared to the control group; §, §§, §§§ significantly different between HE and EX + HE groups; #, ##, ### significantly different between HE and EX group; $, $$, $$$ significantly different between EX + HE and EX group (P < 0.05, P < 0.01, P < 0.001, respectively). CTL = Control; HE = Heat Exposure; EX = Exercise.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Effects of heat exposure and exercise on global oxidative stress. Data are presented as means ± SD. \*, \*\*, \*\*\* significantly different compared to the control group; \$\$, \$\$\$ significantly different between HE and EX + HE groups; \#\*, \*\*, \*\*\* significantly different between HE and EX group; \$\$, \$\$\$ significantly different between EX + HE and EX group (P < 0.05, P < 0.01, P < 0.001, respectively). CTL = Control; HE = Heat Exposure; EX = Exercise.</figcaption></figure>

increase in its activity reveals a pro-inflammatory environment, which is found following acute exercise (Ho et al., 2005). Interestingly, in our study, P-NFkB was significantly more increased in EX + HE than in EX, evidencing that combination of both stressors should intensify inflammatory signaling. Finally, P-DRP1, a mitochondrial fission and potential mitochondrial stress marker, also tends to increase only in EX + HE, suggesting a potential increase in mitochondrial damage. Taken together, these findings suggest that the combination of high-intensity exercise and heat generates a higher cellular stress than either stimulus applied alone. However, these interpretations should be taken with caution since global oxidative stress, measured indirectly via protein carbonyl quantification, was not different between groups at this time point. This suggests that our exercise protocol and heat did not lead to enhanced global oxidative stress at 4-h post-exercise. These findings are consistent with previous data showing no increase in protein carbonyl content following acute exhaustive treadmill exercise (Liu et al., 2000). Antioxidant profile, measured by ABTS radical-scavenging capacity and catalase activity, was not increased either 2 and 6 h post-acute exhaustive swimming exercise (Sun et al., 2016). However, Cu-Zn superoxide dismutase activity was increased 2 h post-exercise but returned to baseline values at 6 h post-exercise (Sun et al., 2016), suggesting that oxidative stress is probably increased immediately after acute exhaustive exercise but returns rapidly to baseline values.

### 4.3. Gene expression of ribosome biogenesis and autophagic markers are differentially regulated by HE, EX and EX + HE

Ribosomes, as ribonucleoprotein complexes that translate mRNA into proteins, have a critical function in enhancing the translational capacity of muscle cells, ultimately leading to protein synthesis and

hypertrophy (Chaillou, 2019; Hammarström et al., 2020; Solsona and Sanchez, 2020). Ribosome biogenesis, a key factor in muscle hypertrophy (Roberts et al., 2015; Normand-Gravier et al., 2025) relies on rDNA transcription mediated by POL1RA, which produces rRNA. In this process, the pre-initiation complex (PIC), which includes key factors such as UBF and TIF-1A, is essential for efficient transcription initiation (Mayer and Grummt, 2006; Goodfellow and Zomerdijk, 2013; Figueiredo and McCarthy, 2019). Our results showed higher expression with HE than similar increases observed in EX of two markers of ribosome biogenesis (POL1RA and UBF). Our data are in line with previous research reporting a rise of ribosome biogenesis markers, including POL1RA, TIF-1A and UBF, following RE in humans (Figueiredo et al., 2016). Furthermore, Figueiredo and coworkers highlighted that RE-induced UBF expression was blunted by cold-water immersion after RE (Figueiredo et al., 2016), whereas hot-water immersion post-RE (only the limbs during 20 min at 41 °C) did not allow ribosome biogenesis in rats, despite increased mTOR signaling and c-Myc mRNA (Kotani et al., 2024). The authors observed a decrease in 28 S and 18 S rRNA content and no effect on 45 S pre-rRNA content and ribosomal RNA and protein expression (Kotani et al., 2024). Consistent with these findings, we found a decrease in 28 S rRNA in all the experimental conditions and a decrease in 18 S rRNA for HE and EX + HE, suggesting that the activation of autophagy (at the protein level for EX and EX + HE and at the gene level for HE) could potentially explain the decrease in ribosomal content observed in all experimental conditions, by selectively degrading ribosomes (i.e. ribophagy) in the early phase of recovery, as observed during cellular stress such as starvation (Kraft et al., 2008). However, in our study and contrary to the findings of (Kotani et al., 2024), gene expression of 45 S pre-rRNA, the precursor of mature 18 S, 28 S and 5.8 S rRNA, was increased for EX and EX + HE, suggesting that our hybrid exercise protocol is a sufficient stimulus to promote ribosomal transcription. However, post-exercise HE did not promote additional effects on this pathway and ribosomal content (as observed with 18 S and 28 S rRNA) was decreased. It can be also hypothesized that the differences observed between 45 S pre-rRNA and mature rRNA 18 S and 28 S expression are linked to rRNA maturation defect upon heat stress (Darriere et al., 2022). However, HE alone was not a sufficient stimulus to increase 45 S pre-rRNA, even if ribosome biogenesis markers (POL1RA and UBF) were increased at the mRNA level. Furthermore, these markers were upregulated to the same extent by HE as by EX. Taken together, these results suggest that high-intensity exercise promotes ribosome biogenesis and increases ribosomal transcription, as observed with 45 S pre-rRNA, but post-exercise HE does not potentiate this effect and HE increases gene expression of ribosome biogenesis markers without an associated increase on 45 S pre-rRNA, evidencing that exercise represents a more potent stimulus than HE on this signaling pathway.

Autophagy, by removing damaged cellular organelles, is a key cellular pathway to maintain cellular homeostasis under stressful conditions, such as endurance exercise or HE (Sanchez et al., 2014; Møller et al., 2019). The current data showed an increase in two autophagic markers (ATG2 and ULK2) with HE and similar increases were observed following EX. Moreover, HE increased a key mitophagy marker (PAR-KIN) and was also significantly increased compared to EX and EX + HE. This upregulation of this marker can facilitate the efficient degradation of deficient mitochondria (Gouspillou et al., 2018; Singh et al., 2024). Another study highlighted that mRNA expression of mitophagy (PAR-KIN) and autophagy (P62 and LC3) markers was increased following acute exhaustive treadmill running session in mice (Vainshtein et al., 2015). Additionally, it was strongly suggested that exhaustive exercise resulted in an increase of autophagic markers (i.e., Beclin, Bnip3 and LC3-B II) and mRNA levels of atrophic markers (i.e., Atrogin-1 and MuRF1) (Zhang et al., 2017). Here, we used a low-volume high-intensity approach for EX group, and high-intensity might compensate low-volume, which can explain the EX-induced increase of autophagic markers. Collectively, our findings suggest that acute HE stimulates both
ribosome biogenesis and autophagy markers to the same extent as EX. Both stressors stimulate these pathways and might be beneficial for the proper removal of damaged organelles following acute environmental or physical stressors.

It is noteworthy that in our study, post-exercise HE inhibited the exercise-induced upregulation of ribosome biogenesis and autophagy markers at the mRNA level. Tamura and colleagues highlighted that acute post-exercise HE significantly increases plasma corticosterone levels more than EX or HE alone (Tamura et al., 2014). Corticosterone is a well-established biomarker of the systemic stress response (Tamura et al., 2014) and it is conceivable that, in our study, the level of plasma corticosterone for EX + HE condition was excessively elevated. This sustained systemic stress response should have led to a decrease in mRNA content associated with ribosome biogenesis and autophagy. However, given the fact that EX + HE increased autophagic flux and microautophagy markers, with an increase in both LC3-B II/I and LAMP2A, it is possible that a feedback mechanism exists. Indeed, an increase of autophagic flux at the protein level can inhibit the transcriptional activation of autophagy and ribosome biogenesis pathways (Zhao et al., 2022).

### 4.4. Gene expression of heat shock proteins and PGC1-α are differentially regulated between HE and EX

HSP70 and HSP90 are heat shock proteins sensitive to thermal changes and their expression is increased following acute kaumatherapy (Goto et al., 2011; Ihsan et al., 2019; Normand-Gravier et al., 2025). These proteins act as molecular chaperones, protecting muscle cells from stress-induced protein damage and preserving cellular function (Dahiya and Buchner, 2019). Consistent with these functions, we observed increased HSP70 and HSP90 across all the experimental conditions, with significantly higher levels in HE and EX + HE compared to EX alone. Our data are in line with other studies reporting that 1 h of passive heat treatment significantly increased HSP70 and HSP90 mRNA expression (Ihsan et al., 2020). Similarly, heating lower body induced a similar response (Kuhlenhoelter et al., 2016). However, at the protein level, HSP70 was not modified and HSP27 was even downregulated for EX at this time point. As HSP27 is a negative regulator of NFkB (Dodd et al., 2009) and P-NFkB is increased in the EX and EX + HE groups, it can be hypothesized that the reduction of HSP27 favors muscle inflammation at the early phase of recovery. PGC1-α, a critical actor involved in mitochondrial biogenesis and function (Halling and Pilegaard, 2020), was found to be activated by both exercise (Edgett et al., 2013) and heat stress (Liu and Brooks, 2012). We also found an increase in PGC1-α mRNA expression in all experimental conditions. However, EX resulted in a significantly greater upregulation compared to HE and EX + HE, suggesting that contrary to the pattern observed for HSPs, PGC1-α is more sensitive to EX than HE. Moreover, NRF2, an activator of PGC1-α (Merry and Ristow, 2016), was also increased by HE and EX, which is consistent with previous findings (Merry and Ristow, 2016; Ihsan et al., 2020). VEGF (vascular endothelial growth factor), an important modulator of angiogenesis (Ferrara, 1999), is another marker whose mRNA expression can increase after heat stress (Kuhlenhoelter et al., 2016) or acute RE (Gavin et al., 2007). However, in the present study, no increase in mRNA expression of VEGF was detected with EX, HE or EX + HE. This lack of response may be due to the timing of muscle sampling, which might have missed the peak expression window.

### 4.5. Limits and perspectives

Even if kaumatherapy can induce similar physiological adaptations as exercise in sedentary or injured people (Méline et al., 2017; Rodrigues et al., 2020), recent studies suggest that exercise represents a more potent stimulus to increase angiogenesis, aerobic metabolism and lipid oxidation (Marchant et al., 2022; Watanabe et al., 2024; Kaluhiokalani et al., 2024). Our study also suggests that GPT represents a more potent

stimulus than HE to promote changes in pathways involved in protein synthesis. However, HE increased mRNA expression of ribosome biogenesis and autophagic markers to the same extent as EX, and the absence of a corresponding increase in protein expression for P-MTOR, P-RPS6 and P-4E-BP1 could be explained by differences in kinetics of protein response. Finally, the addition of HE to EX did not amplify protein synthesis markers but increased some autophagic markers (LC3-B II/I, ATG7, LAMP2A) and P-NFkB, suggesting an increased cellular stress with combined interventions. An optimal dose must be determined to promote skeletal muscle adaptations and avoid potential maladaptation to exercise. Future studies should more precisely examine the kinetics of the two main downstream effectors of MTORC1 (i.e. P-S6K1 and P-4E-BP1) and autophagic flux following different exercise protocols (including hybrid protocols) combined with different heat exposures (i.e. temperature, duration) to establish the optimal dose for these interventions. Importantly, future studies should systematically compare sex-specific responses. In this pilot study, we exclusively used male animals due to documented differences in autophagy responses based on biological sex and environmental stressors such as exercise (Triolo et al., 2022). Specifically, young female mice exhibit higher baseline levels of autophagy-related proteins (including autophagic, mitophagic, and lysosomal markers) compared to age-matched males. Sedentary young females also demonstrate superior autophagosome turnover indices relative to their male counterparts. Notably, exhaustive exercise induced autophagic clearance only in young male mice. A comprehensive investigation is required to delineate sex-dependent mechanistic differences in stress-induced autophagy regulation.

### 5. Conclusion

Our study is the first to highlight that a single bout of heat exposure (HE) increases ribosome biogenesis markers (i.e. POL1RA, UBF), as well as autophagy markers (i.e. ATG2, ULK2), to the same extent as exercise (EX). However, combination of both stimuli blunted these signaling pathways and increased autophagy, as evidenced by a rise of LC3-B II/I ratio and P-NFkB, indicative of heightened cellular stress. Finally, only EX and EX + HE (with no significant difference compared to EX alone) activated MTORC1 signaling. This was evidenced by increased purmycin incorporation and activation of MTORC1 downstream targets, confirming that exercise-not HE-serves as the primary driver of enhanced protein synthesis at the 4-h post-exercise time point. Ribosomal transcription (pre-rRNA 45 S) was also increased in EX and post-exercise HE had no additional effects. However, HE alone did not enhance protein synthesis flux neither ribosomal content (rRNA 18 S and 28 S) nor ribosomal transcription (pre-rRNA 45 S), even if ribosome biogenesis markers were increased at the mRNA level. Further research is needed to determine whether repeated combination of EX and HE confers beneficial or detrimental effects on mature rRNA stability, muscle mass and function. This investigation is warranted because some markers of cellular stress demonstrated a more robust increase compared to isolated interventions. While HE may be promising as an adjunct to exercise for muscle adaptation, studies remain necessary to set up optimal "doses" of post-exercise HE to promote benefits on muscle function and maximize training adaptations. This research axis appears also relevant in the fight against muscle atrophy associated with aging and other deleterious conditions.

### CRediT authorship contribution statement

**Tom Normand-Gravier:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Robert Solsona:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Flavie Arnould:** Visualization, Validation,
**Methodology, Investigation, Formal analysis, Data curation. Roméo Deriaz:** Data curation, Investigation, Methodology, Validation, Visualization. **Christelle Bertrand-Gaday:** Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Fabio Borrani:** Writing - review & editing, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Henri Bernardi:** Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Funding acquisition, Resources. **Anthony M.J. Sanchez:** Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Funding acquisition.

## Data accessibility statement

The datasets used and/or analysed during the present study will be publicly available online (link for downloading all original data from the study (qPCR and Western Blot)) after the acceptance of the manuscript.

## Funding

A "Bonus qualité recherche" from the University of Perpignan Via Domitia (France) and a grant from the Fédération de Recherche Environnement (FEDFREE) were obtained for this study. No external funding was perceived. Open access funding was provided by the University of Lausanne.

## Declaration of competing interest

The authors confirm that this article has no financial or non-financial interests that are directly or indirectly related to the work submitted for publication.

## Data availability

No

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