Acute exercise in elite rugby players increases the ...

Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 492–499

ORIGINAL ARTICLE

Scand J Clin Lab Invest Downloaded from informahealthcare.com by Universita Statale Milano on 10/06/14 For personal use only.

Acute exercise in elite rugby players increases the circulating level of the cardiovascular biomarker GDF-15

EMANUELA GALLIERA1,2, GIOVANNI LOMBARDI2, MONICA G. MARAZZI3, DALILA GRASSO2, ELENA VIANELLO3, ROBERTO POZZONI2 GIUSEPPE BANFI2,3 & MASSIMILIANO M. CORSI ROMANELLI3,4 1Department

of Biomedical, Surgical and Oral Science, University of Milan, Milan, 2Galeazzi Orthopaedic Institute I.R.C.C.S., Milan, 3Department of Biomedical Sciences for Health, University of Milan, Milan, and 4Operative Unit of Laboratory Medicine 1, Policlinico San Donato I.R.C.C.S., San Donato Milanese, Italy Abstract Background. Intense training can lead to a pathophysiological change in serum concentration of a variety of biomarkers. Traditional biomarkers of cardiac injury are very useful in monitoring CVD patients, but in healthy subjects or athletes they cannot be informative enough about the cardiovascular risk, because in these cases their serum levels do not increase over the pathological limit. Therefore novel cardiovascular biomarkers are required in order to allow a better monitoring of sport performance, prediction of overtraining and diagnosis of sport-related cardiac injuries. Growth differentiation factor-15 (GDF-15) is emerging as a powerful cardiovascular injury risk indicator. In this study we investigate the effect of intense physical training of on the circulating levels of GDF-15 in rugby professional players. Methods. Serum GDF-15, Erythropoietin, IL-6, the cardiovascular parameter ST-2, NT-proBNP and routine hematological parameters were measured in a group of 30 rugby players before and after a session of intense training. Results. While ST-2, IL-6 and hsCRP displayed no significant changes after intense training, NT-proBNP and GDF-15 showed a significant increase, even without reaching the pathological level. Discussion. The measure of GDF-15 in professional rugby players could be a useful tool to monitoring their cardiovascular status during training and competition session in order to prevent the onset of collateral cardiovascular adverse event due to the intense training and, in the case of cardiac injury, it could possibly allow a very early diagnosis at the beginning of the pathogenic process. Key Words: GDF-15, cardiovascular risk, rugby, intense training

Introduction It is well recognized that a daily routine physical activity is beneficial in the prevention of many diseases, especially cardiovascular ones (CVD). Nevertheless, in the case of professional endurance athletes (cycling or long-distance running), or impact sports (football and rugby), an intense physical training can have an adverse effect on cardiac functions. Indeed, several endurance exercises have been associated with increase of skeletal muscle, liver and cardiac biomarkers [1–4]. Exhaustive physical exercise leads to an increase of reactive oxygen species, thus resulting in an oxidative stress causing cell injury [5,6]. As a consequence, intense training can lead to a

pathophysiological change in serum concentration of a variety of biomarkers. This phenomenon has been extensively studied in professional football players [7–9] who experienced a significant increase of CVD risk factor. Conversely, a similar sport such as rugby, has been extensively studied for muscle-skeletal injuries and traumatic events, but little is known about professional rugby players’ cardiovascular risk. Traditional biomarkers of cardiac injury, such as troponins or natriuretic peptides (NT-proBNP) are very useful in monitoring CVD patients, but in healthy subjects or athletes they cannot be informative enough about the cardiovascular risk, since their serum levels usually increase but not over the

Correspondence: Emanuela Galliera, Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Università degli Studi di Milano, Via Luigi Mangiagalli 31, I-20133 Milano, Italia. Tel: ⫹ 39 02 5031 5354. Fax: ⫹ 39 02 5031 5338. E-mail: [email protected] (Received 21 October 2013 ; accepted 2 February 2014 ) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2014.905697


GDF-15: cardiac marker in rugby players pathological limit. For this reason, novel cardiovascular biomarkers are required to allow better monitoring of sport performance, prediction of overtraining and diagnosis of sport-related cardiac injuries. Recently, several new cardiovascular biomarkers have been described and applied to the diagnosis and clinical monitoring of cardiovascular risk, both in CVD patients and healthy subjects [10]. Among these molecules, growth differentiation factor-15 (GDF-15), is emerging as a powerful cardiovascular injury risk indicator in CDV patients and also in the general population [11–14]. The cytokine GDF-15, also known as macrophage inhibitory factor-1 (MIC-1), belongs to the transforming growth factor-beta (TGF-β) super family, is secreted at low concentrations from most tissues and it is overexpressed in cardiomyocytes and other vascular cell types under stressful conditions [15]. Recent evidences demonstrated that GDF-15 serum concentration is increased across a wide spectrum of cardiovascular disease and represents an independent prognostic factor; thus, it can be a powerful predictor of mortality for cardiovascular events. GDF-15 is also involved in several regulatory mechanisms, such as inflammation, by regulating IL-6 production [16,17], erythropoiesis and iron homeostasis [18]. In this study we focused on a group of professional rugby players in order to investigate the effect of intense physical training on the circulating levels of GDF-15, and GDF-15-dependent biomarkers, such as erythropoietin, interleukin 6 (IL-6), and canonical hematological parameters. In order to better evaluate the cardiovascular status of the group of professional rugby players, an additional biomarkers of cardiovascular risk, ST-2 and NT-proBNP, was evaluated and compared with GDF-15. ST2 is a member of interleukin 1 receptor family. The marker is linked with cardiac tissue fibrosis consequent to necrosis [19] and with mortality after myocardial infarction.

Materials and methods Subjects Professional rugby players (n ⫽ 29) from the Italian National Team were recruited. All were participating in the 2011 summer training camp held at the end of the competitive season and before the World Championship, from July 4 to August 8. The mean age of the athletes was 27.7 ⫾ 3.9 years. Mean weight was 99.6 ⫾ 12.3 kg at the start and 100.7 ⫾ 11.6 kg at the end of the camp; and the pre- and post mean body mass index ([BMI] weight in kilograms divided by the square of the height in meters) were, respectively, 28.5 ⫾ 2.6 kg/m2 and 28.9 ⫾ 2.4 kg/m2. The study was approved by the reference ethical review board (ASL Milano 1), in accordance with

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according to the principles expressed in the Declaration of Helsinki. Blood samplings were part of the routine control decided by the team medical board and no further blood was requested for the current study. All the subjects involved in this study gave their written consent for data analysis. Dietary and training regimens No drugs or supplements influencing the iron metabolism were taken by athletes. Supplementations were performed, before and after the training, with protein- and amino acid-based beverages and bars. The camp was scheduled after the end of the Italian league and just before the world championship. During the 10 days before the camp, the athletes were under total rest. Training protocol was scheduled on a seven-day basis and consisted of two sessions, two hours each, per day. In the first three days, the morning session consisted of: (i) Warm-up, (ii) weight training, and (iii) plyometrics; the afternoon session consisted of: (i) warm-up, (ii) rugby skills, and (iii) interval training. On the fourth day, there were two light and easy sessions of interval training. The fifth day was off. On the sixth day there were two sessions of free sport activities while, on the last day, there were scheduled two sessions of slow and long distance exercises. Hematological profile Subjects were submitted to two blood samplings: The first day of the camp (T1) and, after 35 days, the last day (T2). Fasting blood samples were collected in the morning (08:00 h) under standard conditions by antecubital venipuncture on subjects at rest in a sitting position, in Evacuated tubes (BD Vacutainer Systems, Becton-Dickinson, Franklin Lakes, USA) for the hematological tests (BD K2 EDTA 3.5 mL tubes) and in 7 mL plain tubes (BD SSTII Advance) for clinical chemistry parameters. Immediately after blood drawing, the tubes were inverted 10 times and stored in a sealed box at 4°C until arrival at the laboratory. Hemoglobin concentration (Hb – g/L), hematocrit (Ht – %), red blood cells (RBC – 1012/L), reticulocyte percentage (Ret %), immature reticulocyte fraction (IRF – %), absolute and differential counts of white blood cells (103/μL), mean corpuscular volume (MCV – fL), mean corpuscular hemoglobin (MCH – pg), mean corpuscular hemoglobin concentration (MCHC – g/L), red cell distribution width (RDW-CV – %) were performed on a Sysmex XE 2100 (Sysmex, Kobe, Japan). For clinical chemistry evaluation, sera were separated from whole blood samples by centrifugation at 3000 rpm for 10 min. Sera were stored at ⫺70°C until analyses performed in a single batch Iron (Fe umol/L), Transferrin (Tf – g/L), and


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Tf saturation (%), total and fractionated bilirubin (umol/L), highly sensitive C-reactive protein (hsCRP – mg/L) and cortisol (nmol/L) were measured on a Siemens Advia1800 (Siemens, Tarrytown, NY, USA); Ferritin (μg/L) was measured on a Siemens Centaur; soluble Transferrin receptor (sTfR, mg/L) was measured on a Siemens BN Prospec (Siemens). Imprecision of the methods were: ⬍ 1.6% for hematology markers, but ⬍ 4% for Ret % (e-Check lot 10990810-811-812, Sysmex) and ⬍ 4.2% for clinical chemistry ones. Regular calibration and check, by both internal and external quality control schemes, were performed, across the study, for all the instruments. A day-by-day control of imprecision was performed on the Sysmex instrument by using fresh blood during the study, giving an imprecision ⬍ 1.6% for Hb, Ht, RBC indices and WBC. All the academic preanalytical warnings concerning the sample handling and transport were accurately followed [20]. Cardiac frequency was measured by Polar S810i, Polar Electro Oy, Kempele, Finland, while blood pressure was measured by Precisa N, Rudolf Riester GmbH, Jungingen, Germany. Serum GDF-15, Erythropoietin, IL-6, ST-2 and NT-proBNP Concentrations of soluble GDF-15, Erythropoietin (Epo), IL-6, ST-2, in serum were determined by commercially available ELISA assays according to the manufacturers’ instructions (R&D System, Minneapolis, MN, USA). For GDF-15 and ST-2 detection assay, serum samples were diluted 1:10 as recommended by manufacturers in the assay procedures protocol. Sensitivity and imprecision for these assays were: GDF-15: The sensitivity of the test was 4.44 ng/L, intra- (CVw) and inter-assay (CVb) coefficients of variation were 3.7% and 2.5%, respectively; Epo: 0.6 IU/L, CVw and CVb were 3.5% and 4.2%; IL-6 0.7 ng/L, CVw and CVb were 3.1% and 2.7%; ST-2: 2.45 ug/L, CVw and CVb were 4.5% and 5.4%. The reported values were supplied by producers. The reference range of serum concentration, as reported by ELISA manufacturers’ instructions (R&D System, Minneapolis, MN, USA) was 3.3–16.6 IU/L for Epo, 0.45–9.96 ng/L for IL-6, 337–1060 ng/L for GDF-15, 6.7–20.4 ug/L for ST-2, and 29.2–176.4 ng/L for NT-proBNP. NT-proBNP was measured using aVitros system (Johnson & Johnson, Raritan, NJ, USA); the instrument precision (coefficient of variance) was ⫺5%. Statistical analysis Statistical analysis was performed by Graph Pad Prism v5.0 software (Graph Pad Software Inc., La Jolla, CA, USA). Normal distribution of values were

assayed by Kolmogorov-Smirnov normality test. All values in the descriptive analysis are expressed as the mean ⫾ SD, when normally distributed, and as median (5th–95th percentile), when not-parametric. The comparison between pre- and post-camp values was carried on by means of a two-tailed paired t-test, for normal values, or Wilcoxon’s matched paired test, when values were not-normally distributed. Correlation analysis was performed by the two-tailed Pearson correlation test (Spearman’s test for not normally distributed values); the same test was conducted to evaluate the correlation between the trends of the parameter across the time-points. The significance level was set at 0.05.

Results All the results are shown in Table I by comparing measures observed before (T1) and after (T2) the training period. After the training period, while the cardiovascular biomarker ST-2 displayed no significant difference, GDF-15 displayed a statistically significant increase, but it remained below the pathological level. For this reason we analyzed several GDF-15-related parameters, describing inflammatory status, hematological and iron homeostasis status. First of all, the GDF-15-related inflammatory cytokine IL-6 was detected at physiological levels and it displayed no significant differences between the two time-points. Conversely Epo, even remaining into physiological range, showed a little but statistically significant increase. On the other hand, RBC, Hb and the parameters describing the iron status (Iron, Ferritin, sTfR and Tf saturation), displayed no significant change between the two time-points, as described in Table I. Only Tf displayed a statistically very significant decrease after the training period; whilst hsCRP (Table I) did not show any variation, cortisol serum concentration increased across the study. ST-2, another biomarker of cardiovascular disorders, showed very low serum concentrations, into the physiological range, with no significant difference between T1 and T2 (Table I). The NT-proBNP values in the rugby players before and after the training session followed by passive and active recovery are presented in Table I. Similarly to GDF-15, the training session increased NT-proBNP concentrations.The median value before training was 27.1 ⫾ 8.51 ng/L (range 27.2–143.8 ng/L) and 47.7 ⫾ 6.41 ng/L (range 29.2–176.4 ng/L) after training [46]. The cardiovascular monitoring of the subjects includes measure of diastolic and systolic pressure and cardiac frequency before and after the training period. As expected in well trained professional athletes, all these parameters resulted within physiological range and displayed no differences between

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Table I. Principal serum biochemical markers analyzed in the rugby players population, before (T1) and after (T2) intense training. Statistical significant variations between T1 and T2 are reported as p values.


Biomarker GDF-15 (ng/L) ST-2 (ug/L) NT-proBNP (ng/L) IL-6 (ng/L) Erythropoietin (IU/L) hsC-reactive protein (mg/L) Leucocytes (⫻ 109/L) Neutrophils (⫻ 109/L) Lymphocytes (⫻ 109/L) Monocytes (⫻ 109/L) Eosinophils (⫻ 109/L) Basophils (⫻ 109/L) Bilirubin (μmol/L) Total Direct (μmol/L) Indirect (μmol/L) RBC (⫻ 1012/L) Reticolocytes % Hb (g/L) Hct (%) MCV (fl) MCH (pg) MCHC (g/L) RDW-CV% Tf (g/L) Ferritin (μg/L) sTfR (mg/L) Transf Sat % Iron (μmol/L) Cortisol (nmol/L) Platelets (⫻ 109/L) MPV (fl)

T1

T2

p value

152.59 ⫾ 33.40 8.77 ⫾ 4.29 27.1 ⫾ 8.51 1.97 ⫾ 1.19 7.41 ⫾ 1.98 0.65 ⫾ 0.21 6.26 ⫾ 1.13 2.70 2.63 ⫾ 0.62 0.50 0.20 0.03 12.66 3.08 9.41 5.15 ⫾ 0.34 1.05 ⫾ 0.29 154.2 ⫾ 8.2 44.18 ⫾ 2.18 85.93 ⫾ 4.04 30.10 349.2 ⫾ 3.8 12.86 ⫾ 0.38 2.50 ⫾ 0.26 151.80 ⫾ 62.91 1.16 29 17.55 ⫾ 12.82 529.18 ⫾ 179.23 211.90 ⫾ 33.1 10.93 ⫾ 0.97

193.86 ⫾ 47.68 8.907 ⫾ 5.63 47.7 ⫾ 6.41 3.35 ⫾ 2.67 9.13 ⫾ 1.91 0.59 ⫾ 0.20 5.30 ⫾ 0.91 2.40 2.24 ⫾ 0.56 0.50 0.20 0.02 15.74 4.28 11.29 5.21 ⫾ 0.32 10.7 ⫾ 0.23 155.3 ⫾ 3.2 45.92 ⫾ 2.32 87.91 ⫾ 3.97 30.10 339.9 ⫾ 6.0 13.09 ⫾ 0.34 2.33 ⫾ 0.23 155.67 ⫾ 67.17 1.19 32 17.37 ⫾ 12.57 595.58 ⫾ 103.38 222.93 ⫾ 42.4 10.82 ⫾ 0.89

p ⬍ 0.001 ns p ⬍ 0.01 p ⬍ 0.05 p ⬍ 0.001 ns p ⬍ 0.0001 p ⬍ 0.001 p ⬍ 0.0001 ns ns ns p ⬍ 0.05 p ⬍ 0.01 p ⬍ 0.05 ns ns ns p ⬍ 0.0001 p ⬍ 0.0001 ns p ⬍ 0.0001 p ⬍ 0.0001 p ⬍ 0.0001 ns ns ns ns ns ns ns

ns, not significant; hsCRP, high sensitivity C-reactive protein; sTfR, soluble Transferrin receptor; Hb, hemoglobin.

pre and post training measure (Systolic pressure: 120.0 ⫾ 11.1 mmHg and 120.0 ⫾ 10.6 mmHg; Diastolic pressure: 78.6 ⫾ 5.54 mmHg and 77.4 ⫾ 5.4 mmHg; Cardiac frequency at rest: 63.8 ⫾ 9.8 and 63.8 ⫾ 8.3, before and after training, respectively). Among the different hematological parameters analyzed and described in Table I no significant differences were observed for platelet count and mean platelet volume, while a slight but very significant decrease of leukocytes was observed. Bilirubin displayed an evident, significant increase probably due to the so-called sport hemolysis phenomenon. Even if RBC count displayed not significant difference, several RBC-related parameters showed little but significant increase (Hematocrit value, MCV, RDW) or decrease (MCHC). Statistical analysis of linear correlation was performed comparing GDF-15 with hematological and GDF-15 dependent parameters analyzed, and the results are listed in Table II. We remark that GDF-15 showed a weak positive correlation with hsCRP, leukocytes count, total bilirubin and a strong positive correlation with Epo, IL-6, while no correlation with ST-2 and cortisol was observed.

Discussion Physical exercise and training induce modifications of urinary and serum concentrations of several laboratory parameters [21,22]. In particular, high

Table II. Correlation analysis of GDF-15 with the biochemical parameters evaluated in the study population of rugby players after intense training session. Correlation results are reported as r2 value and p value. Correlation GDF-15 with main biochemical parameters GDF-15 (ng/L)

vs. vs. vs. vs. vs. vs. vs. vs. vs.

hsCRP (mg/L) cortisol (nmol/L) Epo IU/L Leu (⫻ 10^9/L) Lym (⫻ 10^9/L Neu (⫻ 10^9/L) BilirubinT ot (μmol/L) Bilirubin Dir (μmol/L) IL-6 (ng/L) ST-2 (ng/L) NT-proBNP (ng/L)

0.692 0.197 0.794 0.902 0.693 0.927 0.0593 0.0902 0.786 0.678 0.841

0.0000 0.0042 0.0024 0.0001 0.0000 0.0001 0.0005 0.0037 0.0021 0.0035 0.0001

hsCRP, High sensitivity C-reactive protein; Epo, Erithropoietin; Leu, leukocytes; Lym, lymphocytes; Neu, neutrophils; Mo, monocytes; Eo, Eosinophils; Ba, Basophils.


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intensity exercise could be associated with cardiac muscle damage, represented by release of some molecules, commonly used for diagnosing and monitoring cardiovascular diseases [23]. However, the increase of serum concentration of cardiac biomarkers in subjects who performed intense exercise does not mean in most cases a real cardiac tissue damage, but the release of these molecules from cardiomyocytes is a consequence of an increase of permeability of membranes, due also to the attack of some oxidant species, typically produced by exercise, or through externalization of some subcellular bodies (blebs) containing the markers. Biomarkers are critical for evaluating the impact of different exercise intensities in sports medicine. Since not all the canonical cardiac markers, such as troponin, display significant changes in exercise, especially in endurance training [24], they cannot be considered the best choice now to monitor the cardiovascular status of athletes. For this reason, it is important to find new cardiac biomarkers to detect any alteration of the cardiovascular status of high level athletes, in order to obtain a better monitor of sport performance and an early diagnosis of sport related cardiac damages. In this context, GDF-15 is an independent prognostic value in predicting cardiovascular risk and adverse outcome, over and above clinical and biochemical markers including troponins, and natriuretic peptides as NT-proBNP; thus, we studied the effect of acute training period on GDF-15 circulating levels in professional rugby players. GDF-15 displayed a clear and significant increase after the training period analyzed, even not reaching pathological values, suggesting that, even in healthy rugby players, without known cardiac diseases, GDF-15 is able to remark the adaptation of the cardiac function to the acute exercise. Since GDF-15 is produced by cardiomyocytes under stress condition [25–27], any increase of the circulating levels of this biomarker can be considered as a sign of cardiovascular stress. In accordance with this result, as transitory heart damage linked to possible harmful consequences for professional athletes and to exercise intensity and duration is indicated by the increases of serum NT-proBNP. NT-proBNP serum level was correlated to GDF-15 serum levels. The results clearly indicated that GDF-15 correlate with NT-proBNP (r2 ⫽ 0.841), indicating the importance of GDF-15 as potential biomarker for monitoring and detect any effects of overtraining which may be harmful to the heart in high-level athletes. In order to better understand the clinical meaning of this significant, but not pathological, increase of GDF-15, we analyzed the molecules regulated by GDF-15. IL-6, one of the main inflammatory cytokines, strictly connected to GDF-15 expression and involved in the cardiovascular effect of inflammation, displayed no significant difference and it

remained into the physiological range. A similar effect was observed for non-athlete subjects involved in endurance activity [28]. The stability of IL-6 indicates that the GDF-15 increase is not so high to promote an inflammatory response inducing cardiovascular damage. Indeed, the acute physical training induced a little but significant decrease of neutrophils and lymphocytes, orchestrating the immune and adaptive immune response, respectively, parallel to a slight increase in cortisol. Exercise training is reported to increase neutrophils count in the first 24 hours after acute exercise, but it is able to reduce neutrophils count in chronic inflammatory conditions, acting as an anti-inflammatory [29]. Similarly, acute physical training induces a transient lymphocytosis immediately after exercise, followed by a decrease of these cells until return to the resting value or even below. These results indicate that intense physical rugby training, as recently demonstrated for other sports, can affect several laboratory parameters, with slight effect on hematological profile and iron parameters and more evident effect on the cardiovascular biomarker GDF-15. Notwithstanding the limited number of subjects studied, the trend towards a post exercise increase of GDF-15 indicates that the production of this molecule is clearly influenced by exercise, thereby representing a potential source of biological variability in the clinical assessment. On the one hand, since we consider healthy subjects, the concentration of circulating GDF-15 did not reach pathological levels, commonly measured in severe cardiovascular conditions like hypertrophic cardiomyopathy and heart failure. On the other hand, GDF-15 showed a clear responsiveness to the cardiovascular stress following intense training. Indeed, it has been reported that moderate exercise training did not alter GDF-15 circulating levels even in patients with coronary artery disease while high levels of GDF-15 were shown in professional athletes and, in particular, increased concentrations of GDF-15 were measured after endurance performance and a football match [30]. Among the various cardiovascular biomarkers, GDF-15 was reported to be the most powerful in predicting cardiac damage risk [11]. Indeed, we measured serum concentration of the C-reactive protein [31], a putative marker of coronary heart disease event. The highly sensitive kits allow the detection of the typically low concentrations of circulating in the absence of CRP an overt infectious or inflammatory episode and, thus, to link CRP variations into injuries going beyond the inflammatory episode (i.e., cardio-coronary damage) [32]. No differences were reported in hsCRP after a 21-day long cycling stage race involving professional cyclists [33] and between well trained subjects and their healthy sedentary counterparts [34–36]. However, exercise associated to cardiac rehabilitation,


GDF-15: cardiac marker in rugby players strongly reduced the hsCRP concentration in coronary heart disease patients [37]. These results, together with those here presented show that physical activity, even when really strong, does not affect hsCRP in healthy subjects whilst it is effective in subjects affected by heart diseases. These findings suggest that, rather than hsCRP and IL-6, GDF-15 is more affected by the cardiomyocytes contractile activity; when this activity is exceeding the threshold the GDF-15 levels could increase above the physiologic limits. In order to better define the diagnostic relevance of GDF-15 circulating level increase, we analyzed in the same athletes another cardiovascular risk biomarker, ST-2. This molecule, known also as the IL-33 receptor [38] has been recently described as a powerful marker of cardiovascular disorders [39] and could provide, if compared to GDF-15, a clear definition of a pathological cardiovascular status. In our athletes, ST-2 was very low, even if in the physiological range, without displaying significant difference after intense training. Accordingly, there is no correlation between GDF-15 and ST-2 serum levels. This result clearly indicates that there is not a pathological process ongoing at the cardiovascular level, but the increase of GDF-15 can be considered a response of cardiomyocytes to an intense stress condition. This finding can also suggest a positive, rather than negative significance of GDF-15 increase. It has been reported, indeed, that, differently from TGF beta and other members of TGF beta family, GDF-15 can exert a cardioprotective effect, particularly in cardiovascular events triggering oxidative stress, ranging from pressure overload to heart failure. In vivo studies in mice models demonstrated that in these conditions GDF-15 can be considered a protective molecule for the heart [40]. GDF-15 is secreted by the erythroid cells during their maturation and suppresses the expression of liver-derived hepcidin, which acts by binding the ferroportin-mediated release of iron in blood by amacrophages, hepatocytes, duodenal enterocytes. Suppression of hepcidin allows greater availability of iron for erythropoiesis [41]. When low concentration of iron linked to transferrin (Fe-Tf) signaling causes a decrease in hepcidin transcription, while high concentration of Fe-Tf increase the hepcidin expression. In the present study, under a controlled and sufficient nutritional intake of iron, no variations in the metal concentration were found, confirming that that GDF-15-mediated hepcidin suppression is relevant only in situations of altered erythropoiesis (i.e. thalassemias) [42]. The increase of Epo, bilirubin and RDW, describing the erythrocytes anysocytosis, seems to be linked to an increase of erythropoiesis, which is not, however, so important to modify Hb. In this scenario, the increase of GDF-15 could be interpreted as a sensitive marker of erythropoiesis

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stimulation, typically found in athletes during intense training and competition periods. In the present case, since the physical activity did not affect erythropoiesis, as demonstrated by the stability of the erythrocytes counts, despite the increase in Epo concentrations, GDF-15 variation are plausibly associated to the cardiomyocytes activity increase. The increase of biochemical parameters as GDF-15 , are commonly used for diagnosing and monitoring cardiovascular accidents in clinical patients, while in professional athletes, who are basically healthy subjects, this increase is not necessarily linked to real tissue damage or necrosis. Thus, it is important to screen various parameters proposed for diagnosing eventual cardiovascular involvement and/or damage in professional athletes to avoid misinterpretations of laboratory results. In contrast, the evaluation of cardiac markers is crucial for diagnosing damages in athletes, although they are rare events in well-controlled healthy subjects. GDF-15 displayed a strong positive correlation with Epo serum levels, but their increase was not sufficient to affect erythropoiesis. Consistent with our observation, studies on anemic patients showed that low amounts of GDF-15 are not sufficient to suppress hepcidin production and, therefore, increase erythropoiesis [43]. Since this is the first study, to the best of our knowledge, evaluating GDF-15 in rugby players, this is a pilot study, whose limit is represented by the limited number of subjects analyzed. Future larger studies will add further insight to the use of serum GDF-15 a marker of cardiovascular risk in professional athletes. In conclusion, the measure of GDF-15 in professional athletes could be a useful tool to monitoring their cardiovascular status during training and competition session, in order to prevent the onset of possible cardiovascular adverse event due to the intense training, but especially for following the cardiovascular adaptation to the exercise. In fact, the behavior of GDF-15 during intense exercise is similar to this already reported by our group in the same professional athletes for NT-proBNP [44]. Acknowledgements We are indebted to the medical team and managers of the Italian Rugby National Team for the invaluable cooperation (RP is a member of the medical team). We also thank the Cedal laboratory (Gallarate, Italy) which provided hematological and clinical chemistry data. The authors would also acknowledge the Italian Ministero dell’ Istruzione, Università e Ricerca (MIUR) ed il Ministero della Salute for providing funds for this research project. Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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