No effect of acute exercise on the dynamic change in the expression of genes involved in epigenetic modication in professional athletes

Background: The adaptation of the organism to exercise in the context of gene expression prole is an interesting phenomenon. Exercise can change the expression of individual genes due to changes in the degree of DNA methylation, changes in miRNA expression, or through methylation or acetylation of histones. Hypothesis: Acute exercise increases the expression of genes such as HDAC1, DNMT1, and JHDM1D that can affect epigenetic modications in PBMCs. Methods: The aim of this study was to determine whether there was a change in gene expression in the blood cells during acute exercise and after a 1-hour recovery. The transcriptions of genes involved in epigenetic modications (HDAC1, HDAC1 and JHDM1D) were examined in 9 professional athletes at rest, during consecutive stages of a treadmill exercise until exhaustion, and following recovery. Results: No signicant differences in the level of transcript were observed in the course of the experiment in the tested PBMC cells. On the other hand, a signicant (p = 0.007) correlation was observed in the level of the JHDM1D gene transcript and the number of monocytes in the samples obtained after reaching peak exercise intensity, but in the initial samples this correlation was not signicant (p = 0.053). Conclusion: monocytes


Introduction
Gene expression is in uenced by epigenetic modi cations as well as DNA sequences. Both biological and environmental factors induce these modi cations. One of these factors is exercise (acute and chronic) that can alter the expression of individual genes due to changes in the degree of DNA methylation, changes in miRNA expression, or through methylation or acetylation of histones ( A 6-month exercise training increased the level of methylation in adipose tissue (Rönn et al. 2013) and decreased the level of global methylation in peripheral blood mononuclear cells (PBMC) (Dimauro et al. 2016) and muscle tissue (Barrès et al. 2012).
DNA methylation and histone acetylation and their effect on gene expression during single exercise and training were intensively explored. However, relatively little is known about histone methylation and its impact on gene expression during exercise. Some data is available on the effect of acute exercise on histone methyltransferase (HMT), such as protein arginine methyltransferase (PRMT), or the effect of exercise on methylation of speci c regions, such as the H3K36 region. The methyl group can be removed from this region by the demethylases having a JmjC domain, such as the HMT of the JmjC domaincontaining histone demethylation protein 1 group (JHDM1) (Dimauro et al. 2020).
The potential changes in DNA (cytosine-5)-methyltransferase 1 (DNMT1) expression during exercise appear to be interesting. The transcription of DNMT1 in PBMCs is relatively constant and its expression linearly declines with age (Ciccarone et al. 2016). It is of interest to explore the effect of acute exercise on DNMT1 transcriptions. Considering studies that show that interleukin-6 (IL-6) stimulated cells upregulated DNMT1 expression, and the release of IL-6 is observed during exercise and in ammation (Horsburgh et al. 2015), we can expect increased DNMT1 expression during acute exercise.
The same is true for Sirtuin 1 (SIRT1) deacetylase which reduces SIRT1's activity and expression under the in uence of oxidative stress. Then, SIRT1 affects the acetylation of the promoter region of the It may also be interesting to check the expression of some enzymes responsible for epigenetic control in the blood, as most research focused on muscle or fat tissue. In our study, we selected genes with documented expression in blood mononuclear cells (Nawrocki et al. 2015(Nawrocki et al. , 2017 i.e. DNMT1, HDAC1, and JHDM1D genes.
Most studies on athletes focused on muscle and fat cells. The aim of this study was to determine whether there is a change in gene expression in the blood cells during acute exercise and after a 1-hour recovery. We hypothesized that acute exercise increases the expression of genes HDAC1, DNMT1, and JHDM1D that can affect epigenetic modi cations in PBMCs.

Exercise test
Two days before the visit to the laboratory, the athletes reduced the amount and intensity of their training.
All measurements were performed in the morning, about 2 hours after a light breakfast. First, weight and height were measured using the SECA 285 measuring station (SECA GmbH, Hamburg, Germany). Then, subjects performed an incremental treadmill test until exhaustion on the h/p Cosmos Pulsar treadmill (Sports & Medical GmbH, Nussdorf-Traunstein, Germany). After application of the necessary equipment, once on the treadmill, the athlete stood still on the treadmill for 3 minutes to record resting parameters and check the functioning of the measuring system. The initial speed was set at 4 km h −1 and after 3 minutes increased to 8 km h −1 . After that point, the speed of the moving strip was progressively increasing by 2 km h −1 every 3 minutes until voluntary exhaustion. The main part of the test was followed by a 30-minutes recovery phase consisting in walking at a speed of 4 km h −1 for 3 minutes and then sitting for another 27 minutes. During the test, a number of cardiorespiratory variables were measured constantly (breath by breath) using the MetMax 3b-R2 ergospirometer and analyzed using the MetaSoft Studio 5.1.0 software package (Cortex Biophysik GmbH, Leipzig, Germany). Heart rate was measured continuously with the Polar Bluetooth Smart H6 monitor (Polar Electro Oy, Kempele, Finland). The whole measuring system was calibrated according to the manufacturers' instructions. During the measurements, constant ambient temperature was kept at 20-21°C. At the nal stage of the test, maximal oxygen uptake (VȮ 2 max) and the values of accompanying cardiorespiratory variables were determined. VȮ 2 max was considered achieved if at least three of the following criteria were met: (i) a plateau in VȮ 2 despite an increase in speed and minute ventilation; (ii) blood lactate concentration ≥9 mmol l −1 ; (iii) respiratory exchange ratio ≥1.10; and (iv) heart rate ≥95% of the age-predicted maximum heart rate (Edvardsen et al. 2014).

Blood sampling
Blood samples were obtained via peripheral catheter 1.3 x 32 mm (BD Ven on Pro, Becton Dickinson, Helsingborg, Sweden) inserted retrogradely into the antecubital vein which was kept patent with isotonic saline (0.9% NaCl) during the entire procedure. The blood for molecular analysis was collected into tubes with EDTA (S-monovette K3 EDTA, 7.5 mL, Sarstedt, Nümbrecht, Germany). For lactate concentration measurement, lithium heparin was used as an anticoagulant (S-monovette, 2.7 mL KE, Sarstedt, Nümbrecht, Germany). Lactate in whole blood (20 µL) was immediately assayed using the spectrophotometric enzymatic method (Biosen C-line, EKF Diagnostics, Barleben, Germany).
Blood samples were drawn 12 times: at rest (before the test), at the end of each 3-minute stage beginning from the 10 km⋅h −1 , and then during the recovery phase. Detailed timing of blood sampling is presented in Table 1.

Isolation of PBMC and RNA
On the day of blood sampling, PBMCs were isolated using a Ficoll density gradient (Lu et al. 2015). 3 ml of blood was applied gently to 1.5 ml of Ficoll solution (from Sigma) in a 15 ml tube so that the layers did not mix. The samples were centrifuged for 35 minutes at 400 g at room temperature. The resulting PBMCcontaining interphase was transferred to a new tube and washed 3 times in PBS. Washing was performed by adding 2 ml PBS, vortexing, centrifuging for 10 minutes at 340 g and discarding the supernatant. Finally, the pellet in PBS was transferred to a centrifuge tube and subjected to nal centrifugation for 10 minutes at 250g. The obtained pellet was suspended in Trizol (RiboEx® GeneAll) and frozen at -80˚C.
Further RNA was isolated from the obtained PBMCs according to the protocol of the manufacturer of the RiboEX® reagent from point three. The manufacturer allows the storage of the isolated cells in the RiboEX reagent at -80˚C, which takes place in the second step.
The quality and concentration of the obtained RNA were checked on an agarose gel under denaturing conditions and by measuring the absorption spectrum on the NanoDrop apparatus.

RT-PCR and qPCR
The obtained RNA samples were reverse transcribed into cDNA using M-MLV Reverse Transcriptase® (Invitrogen). The reactions were performed according to the manufacturer's protocol. An equal concentration of templates in each reaction was prepared for RT-PCR reactions in the amount of 100 ng RNA. From each performed reaction, 1 µl of cDNA was collected for the pooled standard sample to derive the standard curves.
HDAC1, DNMT1, and JHDM1D were selected as test genes, and Esterase D (ESD) andPorphobilinogen deaminase (PBGD) genes were selected for internal control and as a reference to assess the relative amount. The matrices for these genes are presented in the table.

Statistical analysis
The normality of the distribution was checked with the Shapiro-Wilk test, and then the correlation matrix was used to assess the signi cance of the related data. Statistical analyses were performed using the STATISTICA 13 software.
When analyzing changes in gene expression during incremental exercise, one can see an initial decrease in the amount of transcript of the studied genes, followed by an increase and subsequent uctuations in the level of the transcript. However, taking into account the standard deviations and the coe cients of variation, the changes in expression seem to be more of an individual matter. A potential tendency would have to be con rmed on a larger sample of participants. Available research demonstrated a global decrease or increase in DNA methylation depending on the tissue, suggesting that the proteins responsible for methylation and demethylation change their activity, whether through activating factors or increasing the expression of genes encoding methylases and demethylases (Dimauro et al. 2020).
It was observed in many studies that physical exercise causes hypermethylation of the genome in adipose tissue. In muscle tissue, acute exercise induces hypomethylation of numerous genes responsible for mitochondrial function. Chronic exercise induces genetic adaptation of a muscle through demethylation of genes involved in mitochondrial, lipid and glucose metabolism, muscle growth and angiogenesis (Dimauro et al. 2020). This may demonstrate that exercise stimulates muscle tissue cells to intensify their activity, but the activity of adipose tissue cells is reduced. In contrast, studies on blood cells showed a reduction in global methylation in PBMCs with exercise (Hunter et al. 2019). Despite the epigenetic changes observed in the literature, the results obtained in this study indicate that acute exercise has no effect on the production of DNMT1, HDAC1 and JHDM1D mRNA in PBMC cells. Myoblast determination protein 1 (MyoD) is one of the proteins that affect the development and repair of skeletal muscle. It was shown that it has greater expression in humans after acute exercise than without exercise (Caldow et al. 2015). Also, acute exercise increased MyoD expression before and after a 12-week resistance training (Mal et al. 2001; Kadi et al. 2004).
HDAC1 has the ability to repress MyoD transcription by maintaining deacetylation (Mal et al. 2001). One can also expect that in people who exercise regularly HDAC1 will less interfere with MyoD deacetylation and its transcription in muscle tissue should not increase. In some researches, an increase in HDAC activity in blood after strenuous exercise was observed in obese subjects. The effect of exercise may be linked to epigenetic control of in ammation (Dorneles et al. 2016). Moreover, the effect of acute exercise on the reduction of HDAC2 activity in PBMC (Dorneles et al. 2017) was observed. Our study showed that HDAC1 transcription is not altered by acute exercise.
In the leukocytes of our athletes, an increase can be seen between the value at rest and after intense exercise. The increase in these leukocytes in the same conditions was also observed by other researchers (Dorneles et al. 2016). We could not nd any research on the relationship between the effects of exercise on leukocytes and genes. Also, the correlation between the JHDM1D transcript and the level of monocytes and HDAC1 transcript with level of lymphocytes should be con rmed. Further research should focus on the level of proteins encoded by HDAC1, DNMT1 and JHDM1D genes and their subcellular locations.