Prognostic value of serum soluble ST2 in professional athletes Valor pronóstico de ST2 soluble en suero en deportistas profesionales

Background: The predictive value of serum soluble ST2 (sST2) biomarker for diagnostics of cardiovascular pathologies is still poorly understood as well as the role of psychological stress on the risk of heart disease. Aim: This study aimed at determining the diagnostic significance of the sST2 level in athletes involved in speed-strength sports. In addition, stress as a risk factor for the development of cardiovascular pathology was assessed and analysed as well. Methods: A prospective study on Greco-Roman wrestlers was carried out at the Centre for Sports Medicine and Rehabilitation (Almaty, Republic of Kazakhstan). All participants (n = 30) were males aged 20 to 34 years. The control group consisted of volunteers (VO) (n = 30). Anthropometric and hemodynamic parameters of athletes were studied along with electrocardiography (ECG) and ECG tests. The sST2 level was determined before (BT) and immediately after (AT) training. The stress level was determined using The Perceived Stress Scale10 (PSS-10). Results: The average age of the athletes was 26.57 ± 3.6 years. The total training experience was 14.57 ± 4.02 years. According to the ECG data, minor deviations from the norm (13.3%) and an abnormal ECG (33.3%) were identified. Echo-CG data showed «moderate» and «pronounced changes» in 23.3% and 53.3% of cases, respectively. The sST2 level of VO (337.1 ± 61.8 pg / mL) was lower than that of BT (548.1 ± 32.6 pg / mL) (p d» 0.001). The sST2 level of AT, it was significantly higher (830.01 ± 71.6 pg / mL) than BT (p d» 0.001). The average and high level of stress among athletes was in 43.3% and 56.7% of cases, respectively. Stress increased the likelihood of developing distinctly abnormal ECG (OR = 1.06, 95% CI 1.01–1.08; p = 0.02). The stress level showed a positive correlation with the sST2 level (r = 0.752, p = 0.01). The sST2 concentration and categorical echocardiography data demonstrated a dependent positive correlation (r = 0.6, p = 0.01). Conclusions: Athletes’ sST2 levels exceeded thresholds both before and after training. Moreover, the relationship between an increase in sST2 levels and abnormal ECG abnormalities and a high level of stress in athletes was determined. sST2 concentration was associated with cardio-pulmonary stress triggered by the cumulative exercise dose as well as with lifelong psychological stress. Our findings indicate that the elevated sST2 concentrations in athletes could be used as the predictive value. However, clinical relevance and results validity require further intensive studies.


Introduction
The assessment of specific biomarkers is critical for the prediction of the risk of developing heart disease (Albert, 2011). One of these biomarkers is the ST2 receptor for interleukin 1, also known as the interleukin-1 receptor-like 1 (IL1RL-1) (Pascual-Figal & Januzzi, 2015). Previous studies have demonstrated the importance of determining the level of sST2 as a biomarker of various pathological processes (Kakkar & Lee, 2008). It has been shown that the concentration of sST2 in the blood increases in many inflammatory diseases (Mueller & Jaffe, 2015). For instance, Weinberg et al. (2003) showed that the levels of circulating serum sST2 might be employed for the assessment of patients with chronic severe heart failure.
Taking into account that symptoms of cardiovascular pathology can be hidden due to adaptive mechanisms, heart diseases can develop asymptomatically (Lee, Chandel, & Simon, 2020;Nakamura & Sadoshima, 2018;Shevchenko, Bohmat, Holovko, & Demianenko, 2019). The pathways leading from biomechanical overload to cardiac remodeling are of primary clinical importance, but this process remains not fully defined (Weinberg et al., 2002).
Therefore, there is a need to expand the choice of diagnostic tools for the timely detection of cardiac disorders and the prevention of possible complications (Kovacs & Baggish, 2016). Aengevaeren et al. (2019) showed that the change in the concentration of sST2 in the blood could be used as a predictor of cardiovascular disease in runners (before and after exercise). However, it must be noted that this study did not compare the level of this biomarker in people who are not involved in sports.
Given the fact that there is a different classification of physical activity depending on the type of sport (Garcia, Muñiz, Rodriguez, & Suarez, 2016;Holfelder, Klotzbier, Eisele, & Schott, 2020;Howley, 2001;Schnell, 2019), it can be assumed that changes in the cardiovascular system of athletes may significantly vary (Barbas et al., 2011). In turn, the concentration of sST2 in various types of sports can also differ. In this regard, prolonged physical activity in athletes can trigger an increase in the size of the heart by 10-20% (Sharma, Merghani, & Mont, 2015). Athletes engaged in strength sports develop pure concentric hypertrophy of the left ventricle leading to an increase in both absolute and relative wall thickness and a significant increase in the diameter of the left ventricle (Pluim, Zwinderman, van der Laarse, & van der Wall, 2000). Apart from that, it should be noted that cardiac fibroblasts and cardiomyocytes produce circulating ST2 in response to stress and overload (Weinberg et al., 2002).
In fact, focussing on the development of strength can lead to exceeding the recommended limit of physical activity, and developing possible complications (Kovacs & Baggish, 2016).
Unfortunately, not only excessive physical activity in athletes (Gustafsson, Sagar, & Stenling, 2017) is a risk factor for cardiac disorders. It has been shown that psychological distress during competitions is an important negative factor as well (Villarreal-Angeles, Rodriguez Vela, Tapia Martínez, Gallegos Sanchez, & Moncada-Jimenez, 2021). Hence, the constant fear of failure and loss also potentiates the impact of psychoemotional stress on physical health (Proietti et al., 2011). The negative role of emotional stress as a trigger for the development of acute coronary disorders was described by Wilbert -Lampen et al. (2008). It was demonstrated that the incidence of cardiovascular complications was doubled not only among athletes, but also among spectators (during the sports matches).
It has been shown that stress may be the most important factor, but it is not the only a cause for the development of cardiac complications in people with pre-existing cardiovascular disorders (Rafanelli, Roncuzzi, Ottolini, & Rigatelli, 2007). However, it is worth noting that psycho-emotional stress is still one of the main factors in the development of cardiovascular diseases (Steptoe & Kivimäki, 2012) and damage of cardiomyocytes (Alevizos, Karagkouni, Panagiotidou, Vasiadi, & Theoharides, 2014). In this regard, the purpose of this study was to identify the diagnostic significance of the sST2 level in athletes engaged in sports (focused on 'speed-strength'). In addition, the study aimed at the assessment of stress as a risk factor for the development of pathology of the cardiovascular system.

Participants
This is a prospective non-randomized study with the participation of 30 male athletes aged 20-34 years (Greco-Roman wrestling) with an experience of 5 years or more continuous training and competition. The study was conducted on the basis of the Centre for Sports Medicine and Rehabilitation (Almaty, Republic of Kazakhstan). During preparation, athletes adhered to a standard diet in accordance with standard time ranges corresponding to normal competitions.
As a control group (VO), the study included 30 volunteers aged 20-34: young people who are not involved in professional sports.

Ethical issues
The study was approved by the Ethical Committee of Kazakh Medical University of Continuing Education, Almaty, Kazakhstan. All athletes were fully informed about the experimental procedures, the possible risks and benefits associated with their participation in the study.

General information
Depending on their age, the study participants were sub-divided into 3 age categories: 20-24 years old, 25-29 years old and 30-35 years old. The athletes possessed the following official sports qualifications, including Master of Sports (MS), Master of Sports of International Class (MSMK) and Honoured Master of Sports (ZMS). The level of systolic blood pressure (SBP) and diastolic blood pressure (DBP) at rest was determined according to the ACC / AHA guidelines (Drawz et al., 2020). The pulse rate at rest (Alexis, 2010), level of VO 2max (Hawkins, Raven, Snell, Stray-Gundersen, & Levine, 2007)

ECG Data Analysis
Before the ECG study, athletes kept a horizontal position for 5 minutes, to adapt to environmental conditions. The study was carried out in the morning time at an air temperature of at least 22 ° C (Sharma et al., 2017). ECG analysis was performed in 12 leads, recording speed 50 mm / s, in the supine position, using the BTL Cardiopoint ECG C600 (UK) apparatus. ECG data were analysed at the Research Institute of Cardiology and Internal Medicine (Almaty, Kazakhstan) by two independent cardiologists and divided into three subgroups (normal, mildly abnormal, and abnormal) according to a previous study (Koch, Cassel, Linné, Mayer, & Scharhag, 2014).

Echocardiographic Data Analysis
Echocardiography was performed using an ACUSON S1000 ultrasound machine of the HELX Evolution series (Germany). In accordance with the recommendations of the American Society of Echocardiography (Churchill et al., 2020) the following parameters were determined: LV volume, mass, relative wall thickness, mitral flow velocity, tissue Doppler velocity, LAV, LVEF. Left atrial deformity and global longitudinal LV deformity were measured using 2D speckle tracking software (Cameli et al., 2017).

Plasma / Serum IL1RL1 (ST2) Determination
Blood samples were taken on an empty stomach, according to standard protocol rules (Giavarina & Lippi, 2017), from the antecubital vein (10 ml), 30 minutes before exercise (BT) and within two hours after exercise (AT). Blood samples were placed in test tubes (Ayset, Turkey) and centrifuged at 300g for 10 min, then frozen, and stored at -70 0 C for the analysis (Boisot et al., 2008). The sST2 concentration was determined using a commercial human sST2 ELISA kit (Westang Biotech Co., Shanghai, China) according to the manufacturer's instructions (Chen et al., 2018).
It has been shown that an intensive trainings can affect the occurrence of haematological changes in blood ( for example, on the level of haemoglobin, haematocrit) (Santos, 2018). Hence, to calculate changes in plasma volume by determining the levels of haemoglobin and haematocrit (to assess the degree of dehydration), 2 ml of venous blood was additionally taken before and after training for evaluation according to the previously described method (Dill & Costill, 1974).
The analysis was carried out in a certified (ISO 15189-2015) scientific clinical diagnostic laboratory of the Scientific Research Institute of FPM named after B. Atchabarov (Almaty, Kazakhstan).

Characteristic of physical activity
The standard training of the study participants consisted of a set of 3-hour exercises for sequential execution. In addition, daily training included outdoor walking, warm-up for 15 minutes before the main set of exercises. The main routine of physical activity consisted of several types of exercises related to general and special physical training. In terms of the complex of general physical training, exercises were performed on gym machines (1.5 hours), such as a horizontal bar (4 sets x 25 reps), parallel bars (4 sets x 30 reps), and exercises to strengthen the muscles of the back (4 sets x 20 reps), the press (4 sets x 25 reps) and jumping out with a pancake (4 sets x 20 reps). According to the special physical training exercises, the athletes practiced wrestling techniques (1.5 hours).

Evaluation of level of stress
The level of perceived stress and subjective perception of the level of tension in athletes was assessed using the «The Perceived Stress Scale-10» («PSS-10») questionnaire. The PSS-10 questionnaire consists of 10 questions and determines how stressful people think the previous month of their life (Anwer, Manzar, Alghadir, Salahuddin, & Abdul Hameed, 2020). Answer options are scored on a 5-point Likert scale (0-4), with scores as follows: 4: never, 3: rarely, 2: sometimes, 1: often, 0: always. It should be noted that items 4, 5, 7 and 8 were calculated positively. An overall score of 13 corresponds to normal stress levels, and a score of 20 or higher indicates 'high-stress' levels requiring therapeutic intervention (Cohen, 1988).Thus, the results obtained can be interpreted as follows: normal score (0-13), average score (14-20) and high score: (> 20) [33].In our study, we used an adapted version of the PSS-10 questionnaire in Russian language. The choice of version 10 of the PSS questionnaire in comparison with other available versions such as PSS-7 and PSS-14 was justified by the fact that the analysis of previous studies shows higher psychometric properties of the PSS-10 questionnaire (Lee, 2012).

Statistical analysis
Statistical analysis was performed using SPSS Statistics 25 programme (SPSS Inc., Chicago, Illinois, USA). Data normality was verified using the Kolmogorov-Smirnov test and the homogeneity of variances (Levene test) were confirmed (p> .05). Once the normal distribution was confirmed, studying groups were compared using the unpaired and paired Student s t test. The statistical significance was set at p<0.05.
The SBP level was 119.67 ± 7.64 mm Hg, the DBP was 79.50 ± 6.9 mm Hg, the pulse rate was 65.6 ± 9.5 beats / min., the average maximum oxygen uptake was 67.3 ± 4.2 ml / kg / min. In the sports category, the overwhelming majority of athletes' n = 19 (63.3%) were Master of International Sports, n = 9 (30%) were Master of Sports and n = 2 (6.7%) were Honoured Master of Sports.

sST2 level in the blood sample
To determine the level of sST2 expression in blood serum, first the serum sST2 level in VO at rest and in athletes in BT was analysed. The results showed ( Figure  1) that sST2 levels were significantly higher (p d™ 0.001) in the BT 548.1 ± 32.6 pg / mL group than in the VO 337.1 ± 61.8 pg / mL. Serum sST2 level for AT 830.01 ± 71.6 pg / mL was significantly higher than BT (p d™ 0.001) (Figure 2).
Evaluation of the results of perceived stress PSS-10 (Table 4) showed that the absence of athletes with normal stress levels, n = 13 (43.3%) was at an average level and n = 17 (56.7%) experienced a high level of stress. The average score on the stress scale was 24.0 ± 6.7.
In univariate analysis, perceived stress increased the likelihood of prevalence of Distinctly abnormal ECG (OR = 1.06, 95% CI 1.01-1.08; p = 0.02). The assessment of the stress level depending on the three components of the ECG study (normal, with minor deviations and abnormal) indicated that the level of perceived stress was significantly higher in athletes with distinctly abnormal ECG 23.9 ± 1.2 versus 19.7 ± 0.7 ( p = 0.05) in Mildly abnormal and against 17.4 ± 1.2 (p = 0.001) in Normal ECG.
The sST2 concentration and categorical echocardiography data demonstrated a dependent positive correlation (r = 0.6, p = 0.01) The level of the stress showed a positive correlation with the sST2 level (r = 0.752, p = 0.01) (Figure 3).

Discussion
To the best of our knowledge, this is the first study

Figure 2
Analysis of serum sST2 levels in athletes before (BT) (n = 30) and athletes after training (AT) (n = 30). Data presented as median (IQR), p=0.001, BT compared to AT;   on the assessment of the concentration of sST2 as a possible indicator of the development of heart disorders in athletes engaged in strength sports. The results obtained showed a comparatively high level of sST2 in combat athletes before training in comparison with the control group. In addition, we observed an increase in the concentration of sST2 after training. Our data are consistent with previous reports, where sST2 scores were above cut-off values in 48% of professional runners before the start of a marathon and in 92% after a marathon run (Aengevaeren et al., 2019).
It has previously been shown that young athletes under 35 years old, in comparison with their peers who are not athletes, had an increased risk of sudden death due to cardiovascular diseases (Corrado, Basso, Rizzoli, Schiavon, & Thiene, 2003). Adaptive mechanisms that develop during intense physical exertion lead to functional and structural changes in the cardiovascular system (Dickhuth, Röcker, Mayer, König, & Korsten-Reck, 2004). Moreover, the thickness of both the septum and the posterior wall of the heart increases to the same extent as the internal volume (van de Schoor et al., 2016). The ratio of mass to volume and, therefore, the maximum systolic wall stress remains constant, in contrast to the pathological forms of hypertrophy (Dickhuth et al., 2004). These findings indicate the need for dynamic monitoring of the state of the cardiovascular system of athletes.
It should be noted that compared to other biomarkers such as natriuretic peptides and troponins (Aimo et al., 2019;Berg, Ruff, & Morrow, 2021;Cediel et al., 2021;Perrone et al., 2020;Wallentin et al., 2021), sST2 concentration is independent on the age, renal function or body mass index (Dieplinger et al., 2009). These findings were confirmed in our study as well.
The pathophysiological explanation for the increase in the concentration of sST2 in the blood serum can be linked to the susceptibility of the sST2 gene to the mechanical stress in cardiac myocytes (Weinberg et al., 2003). However, the question of whether sST2 is an indicator of structural changes in the myocardium remains controversial. Some researchers have come to the conclusion that the concentration of sST2 in nonischemic heart failure reflects the level of hemodynamic stress, but the pathogenic processes in the myocardium (Broch et al., 2015). In addition, sST2 concentration is dependent on endothelial cells, but not in cardiomyocytes (Demyanets et al., 2013).
Apart from that, the concentration of sST2 level can be high in other various pathologies as well (Socrates et al., 2010;Tajima et al., 2007). However, the concentration of sST2 is associated with cardiac fibrosis, and some experiments have shown that sST2 has been expressed in mechanically stressed cardiomyocytes (Weinberg et al., 2002).
According to the PSS-10 questionnaire, all athletes had a medium or high level of stress, of which 57.6% of athletes with scores above 20 on this scale had indirect indications for therapeutic assistance. Cardiovascular changes caused by stress associated with the secretion of catecholamine are similar to those observed during exercise (Gavrilovic, Spasojevic, & Dronjak, 2012;Hammadah et al., 2017;Sarvasti et al., 2020;Webb, Rosalky, McAllister, Acevedo, & Kamimori, 2017).
In the situation when the heart rate and myocardial contractility increase, peripheral vasoconstriction leads to a significant elevation of arterial pressure (Shephard, 1997). In addition, it is necessary to note the important role of not only activation of the sympathetic nervous system in response to stress factors, but also a decrease in cholinergic activity. It can result in an increase in cortisol levels and a decrease in glucocorticoid sensitivity. In turn, it may lead to vascular complications causing cardiovascular pathologies (Wirtz & von Känel, 2017).
In a previous study, echocardiographic findings did not show any pathological correlation between elevated sST2 levels and left ventricular function (Quick et al., 2015), its diameter or mass, but this relationship was found out in another study (Shah, Chen-Tournoux, Picard, van Kimmenade, & Januzzi, 2009). In the present study, distinctly abnormal changes in the ECG were detected, which had a statistically significant relationship not only with an increase in sST2 concentration (p = 0.01), but also with the level of stress perception (p = 0.01). The results obtained indicate the importance of the sST2 concentration in the diagnosis of the cardiovascular system; however, due to its narrow specificity, it is not possible to assert the full role of increasing the sST2 concentration as a predictor of cardiovascular pathology.
The stress associated with participating in multiple sports tournaments can also potentiate the chances of developing cardiovascular disorders, although this influence is largely indirect (Ogilvie et al., 2016). Therefore, there is a need to pay attention to psychotherapeutic support as one of the important components of preventive measures. Our findings indicate that regular monitoring (biochemical and psychological) of the level of stress in athletes can help to optimize the physical activity in the early stages (González-Boto, Salguero, Tuero, González-Gallego, & Márquez, 2008).

Study limitations
This prospective study has some limitations, including a small study sample. Further randomized controlled studies with a longer observational period of changes in a relatively large sample of athletes are needed to interpret the full mechanism of the development of adaptive processes and structural changes in cardiac tissue during the stress. It should encompass an accurate determination of the relationship between stress and the level of sST2 concentration in sportsmen.

Conclusions
Our data showed that athletes' sST2 levels exceeded thresholds both before and after exercise. Moreover, the relationship of increased sST2 levels with abnormal ECG abnormalities and high-stress levels in athletes was determined. The sST2 concentration is associated with cardiopulmonary stress induced by the cumulative exercise dose as well as psychological stress. The role of elevated sST2 level in athletes under the stress requires further intensive studies.

Funding
This research received no external funding. The work was carried out within the Ph.D. dissertation framework.