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Peptides 57 (2014) 20–30

Contents lists available at ScienceDirect

Peptides

j ourna l ho me pa g e: www.elsev ier .com/ locate /pept ides

ow does stress possibly affect cardiac remodeling?

ejana Popovica,∗, Bosiljka Plecas-Solarovicb, Vesna Pesicb, Milan Petrovica,osiljka Vujisic-Tesica, Bojana Popovicc, Svetlana Ignjatovica, Arsen Ristica,vetozar S. Damjanovicc

Division of Cardiology, Faculty of Medicine, University of Belgrade, Visegradska 26, 11000 Belgrade, SerbiaDepartment of Physiology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, SerbiaDivision of Endocrinology, Faculty of Medicine, University of Belgrade, Dr Subotica 13, 11000 Belgrade, Serbia

r t i c l e i n f o

rticle history:eceived 19 February 2014eceived in revised form 5 April 2014ccepted 7 April 2014vailable online 18 April 2014

eywords:tressdrenocorticotropic hormoneortisoldrenocorticotropic hormone receptorolymorphismeft ventricular remodeling

a b s t r a c t

The aim of this study was to evaluate the predictive value of adrenocorticotropic hormone (ACTH),cortisol and ACTH receptor polymorphism (ACTHRP) for left ventricular (LV) remodeling. Thirty-sixelite male athletes, as chronic stress adaptation models, and twenty sedentary age and sex-machedsubjects emabarked on standard and tissue Doppler echocardiography to assess cardiac parametersat rest. They performed maximal cardiopulmonary test, which was used as an acute stress model.ACTH and cortisol were measured at rest (10 min before test), at beginning, at maximal effort, at 3rdmin of recovery, using radioimmunometric and radioimmunoassey techniques, respectively. Promoterregion of ACTHR gene (18p11.2) was analysed from blood samples using reverse polymerization reac-tion with the analysis of restriction fragment length polimorphisam by SacI restriction enzyme. Normalgenotype was CTC/CTC, heterozygot for ACTHRP CTC/CCC and homozygot CCC/CCC. In all participants,ACTH and cortisol increased during acute stress, whereas in recovery ACTH increased and cortisolremained unchanged. 49/56 examiners manifested CTC/CTC, 7/56 CTC/CCC and 0/56 CCC/CCC. Therewas no difference in ACTHRP frequency between groups (�2

(1) = 0.178, p = 0.67). LV mass (LVM) and LVend-diastolic volume (LVVd) were higher in athletes than in controls (p < 0.01) and lower in CTC/CTCthan in CTC/CCC genotype (219.43 ± 46.59(SD)g vs. 276.34 ± 48.86(SD)g, p = 0.004; 141.24 ± 24.46(SD)ml

vs. 175.29 ± 37.07(SD)ml, p = 0.002; respectively). In all participants, predictors of LVM and LVVd wereACTH at rest (B = −1.00,−0.44; ˇ = −0.30,−0.31; p = 0.026,0.012, respectively) and ACTHRP (B = 56.63,34;ˇ = 0.37,0.40; p = 0.003,0.001, respectively). These results demonstrate that ACTH and ACTHRP stronglypredict cardiac morphology suggesting possible regulatory role of stress system activity and sensitivityin cardiac remodeling.

© 2014 Elsevier Inc. All rights reserved.

Abbreviations: A, left ventricular late diastolic filling velocity; ACTH, adrenocorticotropotropic hormone receptor polymorphism; A duration, late diastolic filling duration; BMI,

xercise test; CCC/CCC, homozygot for adrenocorticotropic hormone receptor polymorphisTC/CTC, normal genotype (absence of adrenocorticotropic hormone receptor polymorphelocity; e′ , average annular left ventricular early diastolic filling velocity; FFM, fat free might ventricle; RV A, right ventricular late diastolic filling velocity; RV E, right ventricularlling velocity; RVs′ , right ventricular lateral annular systolic velocity; s′ , average annulressure; TDI, tissue Doppler imaging; VO2, oxygen uptake; VO2/kg, relative oxygen upta∗ Corresponding author at: Division of Cardiology, Faculty of Medicine, University of Beel.: +381 64 3709684; fax: +381 11 3615630.

E-mail address: dejanapopovic@yahoo.co.uk (D. Popovic).

ttp://dx.doi.org/10.1016/j.peptides.2014.04.006196-9781/© 2014 Elsevier Inc. All rights reserved.

ic hormone; ACTHR, adrenocorticotropic hormone receptor; ACTHRP, adrenocorti-body mass index; BSA, body surface area; BW, body weight; CPET, cardiopulmonarym; CTC/CCC, heterozygot for adrenocorticotropic hormone receptor polymorphism;ism); DBP, diastolic arterial blood pressure; E, left ventricular early diastolic filling

ass; FM, fat mass; LV, left ventricle; LVVd, left ventricular end-diastolic volume; RV, early diastolic filling velocity; RVe′ , right ventricular lateral annular early diastolicar left ventricular systolic velocity; SV, stroke volume; SBP, systolic arterial bloodke.lgrade, Veljka Dugosevica 27g, 11000 Belgrade, Serbia.

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ntroduction

Stress condition increases metabolic demands [43,49,76] andonsequently exposure to stress influences cardiac performance14,15,61]. Hormones have been documented to have a crucial rolen adaptation to stress [43] and they also act in accomodation of car-iac function to stress conditions [14,15,61]. The adaptive changes

n athletes, as chronic stress adaptation models, go in the direc-ion of the cardiac enlargement and the whole body’s improvementbility [15,61]. On the other hand, there is so called “broken heartyndrome”, or stress – induced cardiomyopathy, characterized byemporary enlargement of some parts of the heart which lose theirbility to contract and which is brought on by the heart’s reaction to

serge of stress hormones [57]. In that case, stress adaptive changesxhibit marked variability [62], depending on type of stress, but alson age [32], gender, race, [9], nutrition [4], psychological factors46,71], physical activity and genetic factors [2,16].

Stress system activation implies secretion of hypothalamicorticotropin releasing hormone, which stimulates pituitary proo-iomelanocortin (POMC) secretion [82]. POMC is the precursoreptide of adrenocorticotropic hormone (ACTH), which partiallyegulates cortisol secretion from adrenal glands [23,48]. ACTH andortisol, besides epinephrine and norepinephrine, are the mostmportant stress hormones [1]. There is a correlation of circulat-ng ACTH with lactate plasma level, and increased lactate level isonsidered as marker of peracute stress [78]. Thereby, a certainevel of ACTH is a reliable measure of acute stress, unlike the cor-isol, which is more a measure of chronic stress and has protectiveole [52,78]. It is known that ACTH and cortisol have complex rolen the regulation of carbohydrate, lipid and protein metabolism as

ell as energy homeostasis [13], and also in the regulation of bodyuids and body composition, which are directly related to cardiac

unction [67,68,76]. Furthermore, there are some reports show-ng that ACTH and cortisol are also involved in the regulation oflood pressure and heart rate [67,68,76]. However, cardiac effectsf stress hormones and the mechanisms of their action are still notompletely elucidated [76].

In the past decade it has been demonstrated that genetic fac-ors might play an important role in the morpho-functional cardiachanges due to chronic stress. Thereby, they determine to somextent the increase in the left ventricular (LV) mass and diameter,s well as fractional shortening. For example, there is a corre-ation of DD genotype of anigiotensin – converting enzyme (I/Dolymorhism) and LV hypertrophy in endurance athletes [54]. Fur-hermore, the relation of T allele homozygot of angiotensin gene

235T polymorphism and left ventricular mass (LVM) was shown40]. Additionally, the synergism of anigiotensin – convertingnzyme polymorhism and angiotensin gene M235T polymorphismn the determination of the LVM was predicted [24]. However, therere no data on the association of the ACTH receptor polymorhismACTHRP) and the morpho-functional cardiac adaptive changes.

Considering the fact that the endocrine system plays a key rolen stress situation, including physical activity, which is frequentlysed as a stress model [18,64], the aim of this study was to evaluatehe predictive value of stress hormones, ACTH and cortisol, andCTHRP for the cardiac remodeling in athletes.

aterials and methods

articipants

Participants in this study were 36 elite male athletes and

0 sedentary age and sex-mached subjects. Athletes have beenuccessfully competitive in water polo and wrestling at the inter-ational level for the past five years and have trained intensively

or more than 10 years. They performed combined strength and

s 57 (2014) 20–30 21

endurance training protocols and were in a period of preparationsfor the international competition at the time they were examined.Wrestlers had 9 h of wrestling a week, 4 h of power trainingin the gym so as to improve the explosive strength and 4 h ofhigh intensity running a week. Water polo athletes trained 12 ha week in the pool, with at least 2 km of swimming per eachtraining, and three additional hours a week in the gym wherethey performed both power and endurance exercises. Controlsubjects were not engaged in sporting activities other than atrecreational level (lesser then 2 h a week for the last 10 years).Participants underwent the study after they have been given aninformed consent. They declared any diseases, chast pain, lossof conciousness and risk factors (hypertrophic cardiomyopathy,hypertension, arrhythmias, diabetes, renal diseases, cardiac andother infections, smoking, anabolic steroids usage etc.), whichwere the exclusion criteria because of the influence on myocardialfunction and total functional capacity of the body. There was nofamily history of hypertrophic cardiomyopathy and sudden cardiacdeath. Physical examination and blood tests showed that all ofthem were healthy and normotensive. ECG was physiological. Thestudy was approved by the Local Ethical Committee.

Anthropometry

Tanita weight (phase sensitive multi-frequency analyzer DataInput GmbH 2000, using software Nutri 3) was used to obtainbody weight, fat mass (FM) and fat free mass (FFM) by bioelec-trical impedance analysis. Body surface area (BSA) was calculatedaccording to the Du Bois and Du Bois formula [25].

Ergospirometry

Participants embarked on progressive continuous cardiopul-monary exercise test on treadmill based on breath-by-breathmethod to obtain peak oxygen consumption (peakVO2), as themeasure of the functional capacity. The testing of all subjects wasperformed at the same time of the day. The protocol was made bypretesting nine randomly chosen subjects, to optimize the durationof the test (8–12 min) as recommended. It involved 3 min rest, 2 minat speed 6 km/h and 2% inclination, 2 min at speed 9 km/h and 2%inclination, with an increase of inclination for 2% every 2 min after,until the criteria for maximal test were reached, and 3 min recovery[34,83].

Echocardiography

Two-dimensional, M-mode, pulsed Doppler and Tissue Dopplerechocardiography were performed on a Sequoia 512 ultrasounddevice with a 2.5 MHz transducer. Echocardiograms were obtainedby two experienced readers according to criteria of the AmericanSociety of Echocardiography [31,44,55,70] and met standard crite-ria of the technical quality. Echosonographer had no knowledgeof the study participants background. Three to five consecutivebeats during quiet respiration were used for calculation of theDoppler variables in apical four-chamber views with standardtransducer positions. Using this method early (E), late (A) diastolicfilling velocities and A duration of both ventricles were obtained.M-mode echocardiography was performed to assess wall thick-ness and cavity dimension. Left ventricular end-diastolic volume(LVVd) was derived from LV internal dimensions by Teicholz’sformula. Left ventricular mass was calculated from Penn-cubeformula: LVM (g) = 1.04 [(LVDd + IVSd + PWTd)3 − LVDd] − 13.6,

where LVDd is left ventricular end-diastolic diameter, IVSd inter-ventricular septum thickness in diastole and PWTd posteriorwall thickness in diastole [20]. Circumferentional end-systolicwall stress was calculated as: WS (g/m2) = [SBP × 0.5 LVDs2

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reach statistical significance (p = 0.072). Besides, the magnitude ofchanges in ACTH plasma level between phases of experiment inathletes and controls were not different (p > 0.05), which indicatessimilar response.

p=0.027

p=0.044

p=0.02 6

p=0.07 2, ns

2 D. Popovic et al. / P

1 + [(0.5 LVDs + PWTs)2/(0.5 LVDs + 0.5 PWTs)2]}]/[(0.5 LVDs +WTs)2 − 0.5 LVDs2], where SBP is systolic blood pressure, LVDseft ventricular end-systolic diameter, PWTs systolic posterior wallhickness diastole [30]. Tissue Doppler imaging (TDI) was recordedn apical four chamber view during end – expiration at sweeppeed of 50 mm/s. Doppler signal angle was less than 25%. Sampleolume was positioned at 1 cm within the septal and lateralnsertion sites of the mitral leaflets and lateral insertion of thericuspid leaflets. Digitally stored loops of tissue Doppler imagingere used for off line calculations of myocardial velocities. Average

alues of left ventricular lateral and septal annular early diastoliclling velocities (e′) and systolic velocities (s′) were assessed tovaluate left ventricular diastolic and systolic function. Lateralight ventricular annular early diastolic filling velocity (RVe′)nd lateral annular right ventricular systolic velocity (RVs′) weressessed to evaluate right ventricular filling and systolic function55,56]. In addition, novel global diastolic index (E/e′)/RVe′ wasetermined, as a good correlate of total functional capacity ofhe body [63]. Reproducibility of measuring annular velocitiesas determined in 10 randomly chosen subjects. Intraobserver

ariability was examined using Bland–Altman analysis. The 95%onfidence limits of a single estimate of the measurements werealculated as 2 × SD/

√2 and reported as percent of the mean value

6]. All obtained echocardiographic variables were adjusted foreart rate [55,56].

lood analysis

Blood was taken in four phases of the test as follows: 20 ml atest, 10 min before CPET (phase 1); 20 ml at the beginning of CPETphase 2); 20 ml at the maximal effort (phase 3) and 20 ml at therd min of recovery (phase 4). Participants were free of food andrink (except water) at least three hours before collecting bloodamples. All blood samples were taken from braunii which waslaced into the patient’s brachial vein before the test in order tovoid hormonal stimulation by needle punctuation. Samples wereentrifuged on 4000 rpm and kept at −80 ◦C. ACTH was measuredsing immunoradiometric method (ELSA-ACTH, CIS BioInterna-ional, Gif-Sur-Yvette Cedex, France with lower sensitivity limit

ng/l). Cortisol was measured by radioimmunoassay (CORT-CT2,IS BioInternational, Gif-Sur-Yvette Cedex, France, with lower sen-itivity limit 4.6 nmol/l). The intra- and interassay coefficient ofariation was lesser then 10% for all assays.

enetic analysis

Promoter region of ACTH receptor (ACTHR) gene (18p11.2)as analysed from blood samples at rest using reverse polymer-

zation reaction (PCR) with the analysis of restriction fragmentength polimorphism (RFLP) by SacI restriction enzyme. PurifiedCR products were afterwords sequenced by ALFExpress II devicesing Thermo Sequenase Cy5 Terminator Cycle Sequencing Kit (GEealthcare). Normal genotype (wild type), which considers absencef ACTHR polymorphisam (ACTHRP), was presented as CTC/CTC.eterozygot for ACTHRP was presented as CTC/CCC and homozygots CCC/CCC.

tatistics

SPSS software (SPSS version 10.0, SPSS Inc., Chicago, IL, USA)as used for statistical analysis. Classic descriptive parameters

ike median for not normally distributed variables, and mean and

tandard deviation for parametric variables were used to expresshe results. The analysis of distribution of the observed variablesas performed by Kolmogorov–Smirnov test in order to apply para-etric analytical methods. The differences between the groups

s 57 (2014) 20–30

were tested by analysis of variance (ANOVA): post hoc multiplegroup comparisons were assessed with Bonferroni’s method andLSD method. Kruskal Wallis nonparametric ANOVA followed bythe Man Whitney test was used for the variables that deviatedfrom normal distribution. Pearson’s correlation test and Spear-man’s rank correlation test were performed to test the correlationsbetween variables. Multiple regression analysis was used to adjustfor body surface area and heart rate when examining the differ-ences between the groups for Doppler measurements and cavitydimensions. The difference was considered significant when a pvalue was lesser than 0.05, and is highly significant when a p valuewas lesser than 0.01.

Results

Clinical, echocardiographic and hormonal analysis of athletes andcontrols

Clinical characteristics of the study groups are presented inTable 1. The participants were similar in age. BW, BSA, BMI andFFM were significantly higher in athletes, whereas FM was similarin both groups. HR and SBP were higher in control population, andpeakVO2 lower.

LVDd, LVVd, LVM and LV SV were higher in athletes than incontrol population as seen in Table 2. Standard echocardiographicdiastolic filling properties of the left and right ventricle were similarin both groups, except LV A duration, which was longer in athletes.TDI revealed lower e′ and higher global diastolic index (E/e′)/RVe′

in athletes, whereas there were no differences in systolic propertiesof both ventricles (Table 2).

In both controls and athletes plasma ACTH significantlyincreased between phases of the test (phase 1 vs. phase 2 p = 0.01;phase 2 vs. phase 3 p < 0.001; phase 3 vs. phase 4 p < 0.001) as seenin Fig. 1. Circulating ACTH was higher in athletes than in controls infirst three phases of the test (p = 0.027; 0.044; 0.026, respectively),whereas in phase 4 it tended to be higher in athletes but it did not

Fig. 1. Circulating levels of ACTH in athletes and controls in four phases of CPET(10 min before the test – phase 1, at beginning of the test – phase 2, at maximaleffort – phase 3, at 3rd min of recovery – phase 4). p values delineate statisticaldifferences among groups at corresponding phase.

D. Popovic et al. / Peptides 57 (2014) 20–30 23

Table 1Baseline characteristics of the study groups.

Parameter Controls (n = 20 males) Athletes (n = 36 males) Controls vs athletes (p)

Age (years) 21.35 ± 2.08 22.21 ± 3.56 nsBW (kg) 78.07 ± 7.25 87.76 ± 10.49 0.001BSA (m2) 1.97 ± 0.10 2.13 ± 0.19 0.001BMI (kg/m2) 24.14 ± 2.02 26.03 ± 2.77 0.009FFM (kg) 67.92 ± 4.31 75.58 ± 7.12 <0.001FM (kg) 10.14 ± 4.31 12.01 ± 6.08 nsHR rest (bpm) 77 ± 11 65 ± 12 <0.001SBP (mm Hg) 121.00 ± 17.67 115.80 ± 10.76 0.026DBP (mm Hg) 79.25 ± 12.17 83.24 ± 6.89 nspeakVO2 (ml/min) 3918.70 ± 415.08 4961.1 ± 578.46 <0.001a

peakVO2/kg (ml/min/kg) 49.52 ± 5.10 57.85 ± 4.93 <0.001a

Results are presented as mean ± SD. BW–body weight, BSA – body surface area, BMI–body mass index, FFM – fat free mass, FM – fat mass, HR – heart rate, SBP – systolicarterial blood pressure, DBP – diastolic arterial blood pressure, peakVO2 – peak oxygen uptake, peakVO2/kg – peak relative oxygen uptake.

a Adjusted for BSA.

Table 2Analysis of echocardiographic variables in athletes and controls.

Parameter Controls (n = 20) Athletes (n = 36) pa

LV SV (ml) 87.14 ± 16.23 102.10 ± 17.32 0.002LVDd (cm) 5.18 ± 0.31 5.56 ± 0.47 0.001LVVd (ml) 129.15 ± 17.65 153.72 ± 29.24 0.001LVM (g) 184.98 ± 33.73 248.63 ± 42.28 <0.001E (cm/s) 0.82 ± 0.15 0.78 ± 0.12 nsA (cm/s) 0.47 ± 0.09 0.47 ± 0.13 nsA duration (ms) 123.20 ± 22.74 150.82 ± 38.42 0.005RV E (cm/s) 0.59 ± 0.13 0.57 ± 0.10 nsRV A (cm/s) 0.40 ± 0.14 0.36 ± 0.09 nsRV A duration (ms) 144.5 ± 28.43 157.03 ± 33.77 nse′(cm/s) 22.91 ± 7.19 18.37 ± 5.33 0.008s′ (cm/s) 15.71 ± 4.76 15.59 ± 4.50 nsRVe′ (cm/s) 20.79 ± 6.17 17.48 ± 6.38 0.063RVs′ (cm/s) 16.06 ± 3.79 17.3 ± 4.61 ns(E/e′)/RVe′ 0.25 ± 0.16 0.39 ± 0.32 0.030WS (g/cm2) 105.00 ± 20.72 113.78 ± 22.24 ns

Results are presented as mean ± SD. LV SV – left ventricular stroke volume, LVDd – left ventricular end-diastolic diameter, LVVd – left ventricular end-diastolic volume, LVM– left ventricular mass, E – left ventricular early diastolic filling velocity, A – left ventricular late diastolic filling velocity, RV E – right ventricular early diastolic filling velocity,R nular early diastolic filling velocity, s′ – average left ventricular annular systolic velocity,R right ventricular annular systolic velocity, WS – end-systolic wall stress.

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V A – right ventricular late diastolic filling velocity, e′ – average left ventricular anVe′ – lateral right ventricular annular early diastolic filling velocity, RV s′ – laterala Adjusted for HR and BSA.

We observed significant changes of circulating cortisol duringhe test in both athletes and controls, as seen in Fig. 2. In bothroups circulating cortisol increased from phase 1 to phase 3 (phase

vs. phase 2 p = 0.001; phase 2 vs. phase 3 p < 0.001) and remainedlevated until phase 4 (phase 3 vs. phase 4 p > 0.05). Cortisol levelas lower in athletes than in controls in first three phases of the

est (p = 0.001; 0.001; 0.009, respectively), while in phase 4 tendedo be lower in athletes but it did not reach statistical significancep = 0.054). The changes in cortisol plasma level between the phasesf experiment in athletes and controls were similar (p > 0.05), whichndicates similar response.

Additionally, the ratios of ACTH and cortisol plasma levelsACTH/cortisol) in all four phases of the test were significantlyigher in controls than in athletes (p < 0.001; p < 0.001; p < 0.001;

= 0.004, respectively), as can be seen in Fig. 3. We observed theesponse of ACTH/cortisol ratio of similar pattern (p > 0.05) in boththletes and controls. Thus, in both groups it remained unchangedrom phase 1 to phase 2 (phase 1 vs. phase 2 p > 0.05) and increasedetween phases 2 and 4 (phase 2 vs. phase 3 p < 0.001; phase 3 vs.hase 4 p < 0.001).

enetic analysis

The distribution of single alleles of ACTHR gene promoter regionas in accordance with Hardy–Weinberg equilibrium in all partic-

pants together (�2(1) = 0.25, p > 0.05), also in athletes (�2

(1) = 0.2,

Fig. 2. Circulating levels of cortisol in athletes and controls in four phases of CPET(10 min before the test – phase 1, at beginning of the test – phase 2, at maximal effort– phase 3, at 3rd min of recovery – phase 4). p values delineate statistical differencesamong groups at corresponding phase.

24 D. Popovic et al. / Peptides 57 (2014) 20–30

p<0. 001

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p=0. 004

Fig. 3. ACTH/cortisol ratio in athletes and controls in four phases of CPET (10 minbefore the test – phase 1, at beginning of the test – phase 2, at maximal effort –phase 3, at 3rd min of recovery – phase 4). p values delineate statistical differencesamong groups at corresponding phase.

Table 3The distribution of single alleles of ACTHR gene promoter region.

ACTHR promoterregion genotype

Controls n/N (%) Athletes n/N (%) p

CTC/CTC 18/20 (90%) 31/36 (86.11%) nsCTC/CCC 2/20 (10%) 5/36 (13.89%) nsCCC/CCC 0/20 (0%) 0/36 (0%) ns

The results are expressed as frequency. N – the total number of participants in theg–

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Fig. 4. Circulating levels of ACTH depending on ACTHRP presence in all participantsin four phases of CPET (10 min before the test – phase 1, at beginning of the test –

roup, n – observed count of each genotype, CTC/CTC – normal (wild) type, CTC/CCC heterozygot for ACTHRP, CTC/CCC – homozygot for ACTHRP.

> 0.05) and controls (�2(1) = 0.06, p > 0.05) separately, as seen in

able 3.Considering the fact that there were no homozygots for ACTHRP,

n further analysis all participants were divided in two groups basedn the presence of ACTHRP (group with normal genotype presenteds CTC/CTC and group with ACTHRP heterozygot manifestation pre-ented as CTC/CCC).

linical, echocardiographic and hormonal analysis of ACTHRParriers and noncarriers

There were no differences in BW, BSA, BMI, FFM, FM, SBP,BP, peakVO2 and peakVO2/kg between ACTHRP carriers and nonarriers (p > 0.05), whereas HR at rest was significantly higher inTC/CTC than in CTC/CCC (70 ± 13 bpm vs. 60 ± 9 bpm, p = 0.023).hen athletes were analysed separately, HR tended to be higher in

TC/CTC, but it did not reach statistical significance (66 ± 12 bpms. 57 ± 8 bpm, p > 0.05).

LVDd, LVVd, LVM, LV SV and WS were higher in ACTHRP carriershan in non carriers as seen in Table 4. Standard echocardiographiciastolic filling properties of the left and right ventricle were similar

n both groups, except LV E was lower and LV A duration shortern CTC/CCC. TDI revealed lower global diastolic index (E/e′)/RVe′

n CTC/CCC, whereas there were no differences in e′, RVe′, s′ and′

Vs (Table 4). The differences remained significant for LVDd, LVVd,

VM, WS, A duration and (E/e′)/RVe′ when athletes were analysedeparately (Table 5). Additionally, the differences between athleteson carriers and controls non carriers were significant for LVDd,

phase 2, at maximal effort – phase 3, at 3rd min of recovery – phase 4). p valuesdelineate statistical differences among groups at corresponding phase.

LVVd, LVM, e′, A duration, and (E/e′)/RVe′. When athletes carriersand controls noncarriers were analysed separately, the differencesbetween groups were significant for LV SV, LVDd, LVVd, LVM, WSand MV E (Table 5).

In both CTC/CTC and CTC/CCC plasma ACTH remainedunchanged between phases 1 and 2, and significantly increasedbetween 2 and 4 (phase 1 vs. phase 2 p = 0.09; phase 2 vs. phase3 p < 0.001; phase 3 vs. phase 4 p < 0.001), as seen in Fig. 4. In allparticipants together circulating ACTH was similar in CTC/CTC andCTC/CCC in all phases of the test (p > 0.05). Besides, the changesin ACTH plasma level between phases of experiment in CTC/CTCand CTC/CCC were not different (p > 0.05), which indicates similarresponse (Fig. 4). However, when data obtained in athletes wereanalyzed separately, plasma levels of ACTH at rest were signifi-cantly lower in noncarriers than in carriers (14.53 ± 9.35 ng/l vs.26.32 ± 18.53 ng/l, p = 0.031). Circulating ACTH at the beginning ofthe test, at maximal effort and in recovery phase was not differentbetween carriers and non-carriers in the group of athletes, as wellas the changes of circulating ACTH between the phases of the test(p > 0.05).

We observed significant changes of circulating cortisol duringthe test in both CTC/CTC and CTC/CCC (p < 0.001) as seen in Fig. 5.Circulating cortisol increased from phase 1 to phase 3 (phase 1 vs.phase 2 p = 0.005; phase 2 vs. phase 3 p < 0.001). There was no signif-icant change in circulating cortisol from phase 3 to phase 4 (phase3 vs. phase 4 p > 0.05). Circulating cortisol, analysed in all partici-pants together and athletes separately, did not differ significantlybetween CTC/CTC and CTC/CCC in any phase of the test (p > 0.05)The changes in cortisol plasma levels between the phases of exper-iment in CTC/CTC and CTC/CCC were also similar (p > 0.05), whichindicates similar response (Fig. 5).

Besides, by analyzing all participants together and athletesseparately, significant changes between ACTHRP carriers and non-carriers in ACTH/cortsol ratio were found only in the group ofathletes. ACTH/cortisol ratio was lower in CTC/CTC than in CTC/CCCathletes in the first three phases of the test (at phase 1: 0.06 ± 0.04vs. 0.1 ± 0.07, p = 0.03; at phase 2: 0.05 ± 0.03 vs. 0.1 ± 0.06,

p = 0.009; at phase 3: 0.16 ± 0.14 vs. 0.33 ± 0.25, p = 0.047), whereasthere were no differences in phase 4. In all participants together,and athletes separately, the response of ACTH/cortisol ratio during

D. Popovic et al. / Peptides 57 (2014) 20–30 25

Table 4Analysis of echocardiographic variables in ACTHRP carriers and non carriers.

Parameter CTC/CTC genotype (n = 49) CTC/CCC genotype (n = 7) pa

LV SV (ml) 95.32 ± 16.95 109.77 ± 23.84 0.049LVDd (cm) 5.38 ± 0.40 5.90 ± 0.55 0.003LVVd (ml) 141.24 ± 24.46 175.29 ± 37.07 0.002LVM (g) 219.43 ± 46.59 276.34 ± 48.86 0.004E (cm/s) 0.81 ± 0.13 0.66 ± 0.11 0.003A (cm/s) 0.47 ± 0.12 0.46 ± 0.13 nsA duration (ms) 143.49 ± 37.18 119.14 ± 23.20 0.037RV E (cm/s) 0.58 ± 0.10 0.57 ± 0.19 nsRV A (cm/s) 0.36 ± 0.09 0.44 ± 0.22 nsRV A duration (ms) 151.15 ± 32.53 161.29 ± 35.58 nse′(cm/s) 19.88 ± 6.79 20.61 ± 3.25 nss′ (cm/s) 13.42 ± 3.58 11.46 ± 2.44 0.092RVe′ (cm/s) 18.71 ± 6.67 18.27 ± 5.42 nsRVs′ (cm/s) 17.01 ± 4.39 15.21 ± 2.50 ns(E/e′)/RVe′ 0.32 ± 0.25 0.19 ± 0.07 0.009WS (g/cm2) 108.46 ± 21.19 126.61 ± 23.82 0.041

Results are presented as mean ± SD. CTC/CTC – normal genotype, CTC/CCC – heterozygot for ACTH receptor polymorphism, LV SV – left ventricular stroke volume, LVDd –left ventricular end-diastolic diameter, LVVd – left ventricular end-diastolic volume, LVM – left ventricular mass, E – left ventricular early diastolic filling velocity, A – leftventricular late diastolic filling velocity, RV E – right ventricular early diastolic filling velocity, RV A – right ventricular late diastolic filling velocity, e′ – average left ventricularannular early diastolic filling velocity, s′ – average left ventricular annular systolic velocity, RVe′ – lateral right ventricular annular early diastolic filling velocity, RV s′ – lateralright ventricular annular systolic velocity, WS – end-systolic wall stress.

a Adjusted for HR and BSA.

Table 5Analysis of echocardiographic variables in athletes ACTHRP carriers and non carriers and controls non carriers.

Parameter Controls CTC/CTC(n = 18)

Athletes CTC/CTC(n = 31)

Athletes CTC/CCC(n = 5)

Controls CTC/CTCvs athletes CTC/CTC(pa)

Controls CTC/CTCvs athletesCTC/CCC (pa)

Athletes CTC/CTCvs AthletesCTC/CCC (pa)

LV SV (ml) 85.59 ± 15.57 100.97 ± 15.25 113.26 ± 26.17 0.001 0.006 nsLVDd (cm) 5.15 ± 0.30 5.51 ± 0.40 6.08 ± 0.52 0.001 <0.001 0.007LVVd (ml) 127.37 ± 16.70 149.29 ± 24.83 187.32 ± 35.72 0.001 <0.001 0.005LVM (g) 180.75 ± 30.37 241.90 ± 31.13 297.66 ± 30.23 p < 0.001 <0.001 0.005E (cm/s) 0.85 ± 0.14 0.80 ± 0.12 0.68 ± 0.12 ns 0.034 0.052A (cm/s) 0.45 ± 0.08 0.48 ± 0.14 0.39 ± 0.07 ns ns nsA duration (ms) 122.44 ± 21.59 154.72 ± 40.01 114.8 ± 16.59 0.002 ns 0.029RV E (cm/s) 0.60 ± 0.11 0.57 ± 0.09 0.64 ± 0.17 ns ns nsRV A (cm/s) 0.40 ± 0.08 0.37 ± 0.09 0.33 ± 0.10 ns ns nsRV A duration (ms) 141.50 ± 25.25 157.14 ± 35.42 157.2 ± 0.10 ns ns nse′(cm/s) 20.64 ± 6.47 15.83 ± 5.27 17.54 ± 2.40 0.024 ns nss′ (cm/s) 13.21 ± 3.76 13.38 ± 3.55 11.3 ± 1.87 ns ns nsRVe′ (cm/s) 21.13 ± 5.75 17.09 ± 6.89 18.48 ± 4.37 0.052 ns nsRVs′ (cm/s) 16.29 ± 3.77 17.21 ± 4.79 15.70 ± 1.71 ns ns ns(E/e′)/RVe′ 0.25 ± 0.15 0.43 ± 0.34 0.22 ± 0.06 0.015 ns 0.004WS (g/cm2) 105.41 ± 21.47 110.23 ± 21.17 136.73 ± 18.13 ns 0.007 0.012

Results are presented as mean ± SD. CTC/CTC – normal genotype, CTC/CCC – heterozygot for ACTH receptor polymorphism, LV SV – left ventricular stroke volume, LVDd –left ventricular end-diastolic diameter, LVVd – left ventricular end-diastolic volume, LVM – left ventricular mass, E – left ventricular early diastolic filling velocity, A – leftventricular late diastolic filling velocity, RV E – right ventricular early diastolic filling velocity, RV A – right ventricular late diastolic filling velocity, e′ – average left ventriculara locityr

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nnular early diastolic filling velocity, s′ – average left ventricular annular systolic veight ventricular annular systolic velocity, WS – end-systolic wall stress.

a Adjusted for HR and BSA.

he test was of similar pattern (p > 0.05) in both carriers and non-arriers It remained unchanged from phase 1 to phase 2 (phase 1s. phase 2 p > 0.05), increased between phases 2 and 4 (phase 2 vs.hase 3 p < 0.001; phase 3 vs. phase 4 p < 0.001).

eproducibility of TDI measurements

Interobserver variability was ±2.4% for e′lat, ±2.8% for e′s, ±2.1%or s′lat, ±2.4% for s′s, ±3.1% for RVe′; ±3.9% za RVs′. Intraobserverariability was similar ±2.1% for e′lat, ±2.2% for e′s, ±2.1% for s′lat,2.4% for s′s, ±3.0% for RVe′; ±3.8% for RVs′.

orrelations of hormones and genetic variables withchocardiographic variables

In all participants together, we observed significant correlationsf hormonal variables and cardiac parameters. Circulating ACTH

, RVe′ – lateral right ventricular annular early diastolic filling velocity, RV s′ – lateral

at 1 correlated with LVM (r = −0.30, p = 0.023), LVDd (r = −0.27,p = 0.047), LVVd (r = −0.31, p = 0.02), LV A duration (r = −0.38,p = 0.004) and RV A duration (r = −0.29, p = 0.027). Plasma ACTH at3 linearly correlated with LV A duration (r = −0.36, p = 0.006) and(E/e′)/RVe′ (r = −0.27, p = 0.044), however the relation of ACTH at3 and (E/e′)/RVe′ can be better described by inverse regressioncurve (for 1/ACTH at 3 = 0.50, p < 0.001, according to equationy = 0.27 + 2.13/x), as can be seen in Fig. 6.

Circulating cortisol at phases 2 and 3 correlated with RV A dura-tion (r = −0.42, −0.31; p = 0.01, 0.019, respectively).

ACTH/cortisol ratio at phase 1 correlated with LVDd (r = −0.27,p = 0.048), LVM (r = −0.28, p = 0.042), LV SV (r = −0.31, p = 0.026).ACTH/cortisol ratio at phases 2, 3 and 4 correlated with LV Aduration (r = −0.31, p = 0.02; r = −0.38, p = 0.004; r = −0.31, p = 0.02;

respectively). ACTH/cortisol ratio at phase 3 correlated with RV Aduration (r = −0.27, p = 0.048).

In all participants together, Spearmann’s analysis showed strongcorrelation of ACTHRP with LVM (r = 0.35, p = 0.008), LVVd (r = 0.31,

26 D. Popovic et al. / Peptide

No, n=49Heterozygo t, n= 7

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ACTHR P presenc ein all participants

Fig. 5. Circulating levels of cortisol depending on ACTHRP presence in all partici-pants in four phases of CPET (10 min before the test – phase 1, at beginning of thetest – phase 2, at maximal effort – phase 3, at 3rd min of recovery – phase 4). pvalues delineate statistical differences between groups at corresponding phase.

1,25

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Ve` (

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Fig. 6. Relation of ACTH level at maximal effort and global diastolic index.

= 0.019), LVDd (r = 0.31, p = 0.019), E (r = −0.38, p = 0.004) and LVV (r = 0.26, p = 0.049).

orrelations of anthropometric variables with echocardiographic,enetic variables and hormones

In all participants together, we observed significant correla-ions of anthropometric variables and cardiac parameters. LVVdorrelated with BMI (r = 0.48, p < 0.001), BSA (r = 0.47, p < 0.001),M (r = 0.42, p = 0.001) and FFM (r = 0.60, p < 0.001). LVM corre-ated with BMI (r = 0.56, p < 0.001), BSA (r = 0.49, p < 0.001), FMr = 0.55, p < 0.001) and FFM (r = 0.56, p < 0.001). LV SV correlatedith BMI (r = 0.47, p < 0.001), BSA (r = 0.44, p = 0.001), FM (r = 0.38,

= 0.003) and FFM (r = 0.62, p < 0.001), whereas s′ correlated with

M (r = −0.34, p = 0.01) and FFM (r = 0.37, p = 0.004). Additionally, LV

duration correlated with BSA (r = 0.40, p = 0.002) and FFM (r = 0.44, = 0.001), RV A duration correlated with FM (r = −0.30, p = 0.024)nd FFM (r = 0.35, p = 0.007), and global diastolic index (E/e′)/RVe′

s 57 (2014) 20–30

correlated with BMI (r = 0.29, p = 0.029), FM (r = 0.34, p = 0.009) andFFM (r = −0.37, p = 0.005).

We also observed in all participants together that ACTH at 1 and2 correlated with FFM (r = −0.39, −0.38; p = 0.003, 0.004, respec-tively). Additionally, ACTH at 2 correlated with BMI (r = −0.28,p = 0.037) and BSA (r = −0.32, p = 0.017).

There were no correlations of anthropometric variables withcortisol plasma levels and ACTHRP (p > 0.05).

ACTH/cortisol ratio at phase 1 correlated with FFM (r = −0.29,p = 0.038), at phase 2 correlated with BMI, BSA, FFM (r = −0.29,p = 0.038, r = −0.28, p = 0.039, r = −0.36, p = 0.006; respectively), atphase 3 correlated with FFM (r = −0.30, p = 0.026).

Predictors of echocardiographic variables

The multiple regression analysis, which included hormonal,genetic and anthropometric variables, demonstrated in all partici-pants together that the best independent predictors of LVM were:FFM (B = 3.84, = 0.58, p < 0.001) and ACTHRP (B = 58.31, = 0.39,p < 0.001), according to equation y = −61.05 + 3.84·x1 + 58.31·x2(x1 = FFM, x2 = ACTHRP); r2 = 0.48, SE = 36.75, F = 24.71, p < 0.001.When FFM was excluded from analysis, the best predictors of LVMwere: ACTH level at phase 1 (B = −1.00, = −0.30, p = 0.026) andACTHRP (B = 56.63, = 0.37, p = 0.003); r2 = 0.17, SE = 0.27, F = 5.48,p = 0.007.

Furthermore, the multiple regression analysis demonstratedin all participants together that the best independent pre-dictors of LVVd were: FFM (B = 2.23, = 0.60, p < 0.001) andACTHRP (B = 34.86, = 0.41, p < 0.001), according to equationy = −21.43 + 2.23·x1 + 34.86·x2 (x1 = FFM, x2 = ACTHRP); r2 = 0.52,SE = 19.99, F = 28.64, p < 0.001. When FFM was excluded from anal-ysis, the best predictors of LVVd were: ACTH level at phase1 (B = −0.44, = −0.31, p = 0.012) and ACTHRP (B = 34, = 0.4,p = 0.001); r2 = 0.26, SE = 24.86, F = 9.15, p < 0.001.

Additionally, the same analysis demonstrated in all par-ticipants together that the best independent predictors of s′

were: FM (B = −0.19, = −0.28, p = 0.022) and ACTHRP (B = −2.56, = −0.25, p = 0.046). Also, LV SV was independently predicted by:

FFM (B = 1.51, = 0.63, p < 0.001) and ACTHRP (B = 14.96, = 0.27,p = 0.012); r2 = 0.46, SE = 13.77, F = 22.21, p < 0.001.

In all participants together, global diastolic index (E/e′)/RVe′ wasindependently predicted by FM (B = 0.02, = 0.31, p = 0.015) andACTH at phase 3 (B = −0.001, = −0.29, p = 0.025) according to equa-tion y = 0.21 + 0.02·x1 − 0.001·x2 (x1 = FM, x2 = ACTH at 3); r2 = 0.17,SE = 0.26, F = 5.48, p = 0.007. We also observed that cortisol at phase2 predicted RV A duration (B = −0.11, = −0.42, p = 0.01).

Discussion

The response to physical stress implies signals from nervous,endocrine and immune system, which lead to morpho-functionalchanges in various organ systems [17,76]. In this study, asexpected, athletes showed different cardiopulmonary adaptivechanges compared to control population [15,61]. They exhibitedhigher peakVO2, which is a representative of the functional capac-ity, as a result of higher FFM and cardiac output. Better cardiac workefficiency was achieved due to enlarged cardiac dimensions andlower heart rate according to La Place’s law. Besides, global diastolicadaptive index as a positive correlate of peakVO2 [63] was higherin athletes, as well as the duration of late diastolic filling, whichindicates better compliance of the left ventricle. The differences in

cardiopulmonary parameters between study groups represent theadaptive response to metabolic demands with hormonal regulatoryrole as crucial [43,49,76]. The changes in the organ systems due tostress are likely mediated at least partially trough repeated and

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rolonged activation of HPA axis, which was the first to be respon-ible for adaptation to stress [66]. In order to reduce bias regardingody size, cardiopulmonary parameters were adjusted only forody surface area, leaving the space for analysis of hormonal regu-

ation of body composition, and it’s indirect effects to the heart.In this study both athletes and controls showed the increase

f plasma ACTH and cortisol during acute physical stress. Therere numerous reports demonstrating the relationship betweenhysical stress and hypothalamo-pituitary-adrenal (HPA) axis2,16,18,21,38,58,79,81], but with controversal findings. Sometudies have documented the increase in plasma ACTH level dur-ng acute physical stress, followed by unchanged cortisol [26,47,84]

hile others have shown the increase of both ACTH and corti-ol during dynamic and static physical stress, depending on itsntensity and duration [28,41], which is consistent with our find-ngs. Our results also revealed higher plasma levels of ACTH andower plasma levels of cortisol in athletes in basal conditions anduring acute physical stress comparing to controls, and are inccordance to previous studies which demonstrated that chronichysical stress may alter basal levels of HPA hormones [8,33,50].tatic training of high intensity increases basal cortisol, whereastatic training of low intensity and long duration decreases basalortisol [3,29]. Intensive aerobic training increases basal levels ofoth ACTH [35] and cortisol [8,74]. On the other hand, some stud-

es have shown the increase in basal cortisol level with unchangedCTH in athletes [72,73]. Thus, plasma levels of HPA hormones areependent on type, duration and intensity of stress.

Furthermore, chronic physical stress may alter the sensitivity ofPA-axis [39]. The literature data about HPA axis activation in dif-

erent stress adaptation models are inconclusive. There are datahowing no changes in HPA axis activation in both trained andntrained individuals [42]. Others have demonstrated that only feweeks of training decreased responsiveness of HPA axis [36]. In

hat direction, decreased response of ACTH in physical stress washown in endurance athletes [49], but static training, on the con-rary, may increase cortisol response [29,53]. This was explaineds a protective mechanism in condition of repeated stress in ordero keep the reserves of the body from exhaustion [49]. In thistudy, the ratio of ACTH and cortisol was lower in athletes, butt increased to the same extent from the beggining to the end ofcute stress in both athletes and controls. Thus, the adaptation tohronic physical stress implied lower plasma levels of ACTH neededor the same cortisol response in any particular phase of acute stresshen in unstressed individuals. However, during acute stress ACTHncreased more than cortisol and the increase was of the same mag-itude in both groups. It is obvious that chronic stress altered HPAxis set up by means of lowering ACTH level, but the activationuring acute stress in both stressed and unstressed individuals wasimilar. This finding suggests that in different stress adaptive mod-ls there might be different sensitivity of adrenal glands to ACTHtimulation with possible diverse involvement of other regulatoryechanisms [48]. This theory is supported by morphological and

iochemical changes in adrenal cortex and medulla due to exercise5].

The issue is more complicated as a result of physiological vari-bility of stress system activation [62]. For instance, HPA axisctivity is also genetically determined [75]. In this sense, theensitivity of hormonal receptors has important role [27,60]. Forxample, the absance of adrenal response to ACTH was shownn subjects carrying mutation of gene for ACTH receptor [12,22].ccordingly, there is different sensitivity of ACTH targets afterPA stimulation [26]. It is known that gene polimorphism for

lucocorticoid receptors (BclI RFLP, N363S, ER22/23EK) and miner-locorticoid receptors (MR180V, MR189I) has influence on cortisolomeostasis in stress condition [19,85]. Furthermore, a polymor-hism within the transcription initiation site of the ACTH receptor

s 57 (2014) 20–30 27

promoter altering the consensus sequence from CTC to CCC atposition 2 of 18p11.2 gene is associated to a higher ACTH/cortisolratio after CRH stimulation, suggesting a decreased adrenal respon-siveness to endogenous ACTH [75]. Thus, ACTH receptor promoterpolymorphism results in a lower promoter activity in vitro andis associated with a lower cortisol secretion to prolonged ACTHstimulation in vivo. Bearing all that in mind, we checked the pres-ence of ACTHRP in both controls and athletes. Despite of smallnumber of carriers per group for that kind of investigation, ourresults are in accordance with the presence of ACTHRP in healthymen (80.2% for CTC/CTC, 19.0% for CTC/CCC, and 0.8% for CCC/CCC,respectively) [75]. Although the upword regulation of ACTH recep-tor expression is managed by ACTH itself [59,87]. Slawik et al. [75]did not find the differences between the carriers and noncarriersof ACTHRP in serum concentrations of ACTH and cortisol, prob-ably because of the HPA axis compensatory mechanisms, but itwas shown that the rate of glucocorticoid excretion was higherin CCC homozygots. In addition, ACTH/cortisol ratio was shown tobe higher in CCC carriers in prepubertal children, demonstratingdecreased sensitivity of HPA axis [45]. In this study, serum con-centrations of ACTH and cortisol, and their response during acutestress, were similar in carriers and noncarriers when analysing allparticipants together, but in the group of athletes separately plasmalevels of ACTH at rest were higher in ACTHRP carriers, as well asACTH/cortisol ratio at rest and at maximal stress. It seems thatprolonged repeated activation of stress axis in chronicaly stressedsubjects demarks inherited sensitivity of ACTH receptor leading toconsequent hormonal changes, which may be an explanation fordifferent morpho-functional adaptive changes of the body duringphysical stress.

ACTH receptor is melanocortin 2 receptor – MC2R [86] whichis necessery for the adrenal gland function and streroidogenesisand according to current knowledge it is located in adrenal cortex,skin and adipose tissue [10,11]. There are literature data showingthe relation of ACTH receptor to psychiatric diseases [65,87,88].In this study, ACTHR polimorphism carriers exhibited lower heartrate at rest. Furthermore, left ventricular mass and diameter werehigher in ACTHRP carriers and they also showed improved sys-tolic function and reduced compliance of the left ventricle. Cardiacdifferences in ACTHRP carriers and non carriers were more evi-dent when only athletes were analysed. Left ventricular mass andcavity dimensions were higher in athletes ACTHRP carriers andthey also showed reduced biventricular compliance and increasedleft ventricular end-systolic wall stress. It seems that improve-ment in stroke volume in the group of carriers when both athletesand controls were analysed was a consequence of athletic perfor-mance. Also, the increase in wall stress and the impairment in leftventricular filling in athletic group was probably a cosequence ofACTHRP presence more then physical activity itself. Accordingly,when athletes carriers and controls non carriers were compared,the significance of differences between groups for left ventricu-lar mass, cavity dimensions and left ventricular end-systolic wallstress was higher, indicating aditive effects of physical activity andACTHRP presence. Furthermore, biventricular compliance betweenthese groups was similar, probably because of their oponent efects.These data demontrate that sensitivity of ACTH receptor and con-sequent alteration of HPA axis function influence cardiac adaptivechanges regardless of physical activity habits. Indeed, relevant neg-ative correlations of ACTH and ACTH/cortisol ratio at rest andmaximal effort with left ventricular mass, diameter and biventricu-lar diastolic function were found. Moreover, ACTH at rest negativelycorrelated with fat free mass, as well as ACTH/cortisol ratio at

rest and maximal stress, demonstrating possible regulatory role ofstress axis activity and sensitivity for body composition, which istightly related to cardiac structure and function, as shown in thispaper too. It seems that higher ACTH at rest, as measure of acute

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tress, is correlated with smaller heart and lower FFM. Additionally,he relation of global diastolic index, as a measure of biventricularompliance and positive correlate of VO2peak [62], with ACTH ataximal stress is inverse, indicating that higher stress is relatedith impaired compliance of the left ventricle. Thus, there is com-lex interrelation of stress hormones, cardiac performance andody composition. In the past decade, it was shown that a longerm stress system activation increases risk for obesitas and cardiaciseases [61]. Furthermore, a certain reduction of cortisol plasma

evel and its catabolic effects are necessery for muscle hypertrophyuring chronic physical stress [80]. It is also known that there is reg-latory role of cortisol to metabolic activity of adipose tissue, and

ts hormones leptin and ghrelin, which directly effect myocardium67,68]. All together, stress system might effect heart directly orndirectly by regulation of body composition, which gives us mate-ial to adjudicate positively in terms of our hypothesis.

The fact is that testing variations of the one particular gene forhe evaluation of a complex system which is modulated by a num-er of factors that interact [51], cannot provide the information thatully determinate. The effect of one single factor in that condition

ust be small. However, our results revealed that ACTHR polimor-hism presence is strong positive predictor of left ventricular massnd diameter, as well as systolic function, independently of ACTHlasma level. Besides, ACTH level at rest was shown to be goodegative predictor of left ventricular mass and diameter, proba-ly because of its relation to body composition. Additionally, ACTH

evel at maximal stress negatively predicted global diastolic func-ion of the heart together with fat mass. To our knowledge, thiss the first study to show the relation of ACTH and ACTH receptorolimorphism to cardiac structure and function.

Cardiac hypertrophy is independent predictor of heart failure7], and there are numerous factors affecting its occurrence, butrst of all genetic factors. Even in athletic heart development genet-

cal factors play important role [40,69]. It was already mentionedhat I/D polimorphisam of angiotensine converting ensyme genend M235T polimorphisam of angiotensine gene are related to car-iac hypertrophy in endurance athletes (D/D genotype) [37,40,54],nd some studies demonstrated theirs negative modifying rolen pathological conditions as cardiomyopathies and myocardialnfarction [77]. Furthermore, the polimorphisam of one single gene

as predicted to have a weak relation to cardiac remodeling, butt is possible that combination of genes may have a sinergisticffects [24]. It seems that some genetic combinations of renine,ngiotensine and aldosteron system might be related to increasedorbidity in athletes favorizing pathological hypertrophy [24].

he role of ACTHR polimorphisam in prediction of cardiac morho-unctional changes is novel fact contributing clarifying genetical

echanisms involved in cardiac remodeling due to stress. The con-equent altered activity and sensitivity of HPA axis is related toardiac remodeling directly or indirectly due to its relation to bodyomposition. Thus, it is inappropriate to make simple mechanicalonclusions about cardiac remodeling if we take into considerationomplex genetical and hormonal factors behind.

onclusion

ACTHRP carriers exhibit lower heart rate at rest, higher leftentricular mass and diameter, improved systolic function andeduced compliance. Our results revealed for the first time thatCTHRP strongly predict cardiac morphology and function suggest-

ng possible regulatory role of stress system activity and sensitivity

n cardiac remodeling. Besides, ACTH was shown to have pre-ictive value in the same sense, probably due to its relation toody composition. Thus, cardiac adaptive changes due to stress are

ikely mediated at least partially trough repeated and prolonged

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s 57 (2014) 20–30

activation of HPA axis, which acts in respect of genetical predispo-sition.

Limitation of the study

This is a pilot study, so as our data are preliminary and furtherinvestigations of this topic on a higher number of participants areneeded.

Disclosures

The authors have nothing to disclose.

Acknowledgement

We thank the subjects who participated in this study. Thisresearch was supported by the Ministry of Education, Science andTechnological Development of Serbia (Projects Nos 175036 and175086).

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