**1. Introduction**

Regular physical activity has many beneficial effects with regard to the cardiovascular system: it induces a physiological remodeling of the heart and results in molecular and cellular adaptive changes, which themselves have a cardioprotective effect. The long-term and moderate practice of sports, especially aerobic–isotonic ones, are even encouraged

**Citation:** Costache, A.-D.; Roca, M.; Honceriu, C.; Costache, I.-I.; Leon-Constantin, M.-M.; Mitu, O.; Miftode, R.-S, .; Mas,taleru, A.; Iliescu-Halit,chi, D.; Halit,chi-Iliescu, C.-O.; et al. Cardiopulmonary Exercise Testing and Cardiac Biomarker Measurements in Young Football Players: A Pilot Study. *J. Clin. Med.* **2022**, *11*, 2772. https:// doi.org/10.3390/jcm11102772

Academic Editor: David Rodríguez-Sanz

Received: 15 April 2022 Accepted: 12 May 2022 Published: 14 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in those with chronic cardiac diseases [1]. Even in the older population, regular physical activity has been shown to slow degenerative cardiac changes and significantly improve quality of life [2].

Concerning young athletes, there are both morphological and functional adaptations of the heart in relation to sustained and intense physical activity. Morphologically, the myocardium suffers a process of hypertrophy and the chambers become dilated, as to adapt to the increased hemodynamic stress. The main difference between these physiological adaptative changes and the pathological ones, which are encountered in chronic cardiac diseases, lies in the fact that hemodynamic stress is intermittent in physical activity, whereas in chronic cardiac disease it is continuous. These structural modifications, especially the enlargement of the chambers, together with the increased vagal tonus, are the ones that, in turn, lead to electrophysiological modifications [3].

Most of the electrophysiological changes, in the context of constant physical activity, which have an electrocardiographic (ECG) expression, can be interpreted as within the normal limits without warranting further investigation. However, they are not to be confused with pathological ECG patterns, which have an underlying cardiac pathology and which can act as triggers for adverse events, such as sudden cardiac death [4,5] (see Table 1).


**Table 1.** Electrocardiographic patterns in athletes and their interpretation (RBBB—right bundle branch block; AV—atrio-ventricular; PCV—premature ventricular contraction) (adapted from Sharma et al. [5]).

The 2020 European Society of Cardiology (ESC) guidelines on sports cardiology and exercise in patients with cardiovascular disease are a milestone in both the field of cardiology and sports medicine. As the first edition, they introduce numerous up-to-date information and recommendations for the practice of physical exercise in both healthy individuals and persons with known cardiovascular pathologies. Among the potential adverse cardiovascular events, the two most dangerous are sudden cardiac arrest (SCA) and sudden cardiac death (SCD). As tragic as they may be, especially if occurring in young healthy individuals, SCA and SCD are not reported in all countries; therefore, their incidences cannot be accurately estimated. However, according to most recent statistics, SCA

is diagnosed in 1 out of 80,000 high-school-aged athletes and in 1 out of 50,000 college-aged athletes. Based on gender, ethnicity, and type of sports practiced, the most at-risk group is represented by male, black, basketball athletes in the United States, or football athletes in Europe [6–11] (see Table 2).

**Table 2.** Exercise-related major adverse cardiovascular events (MACE) [6].


The most important etiology of SCD in young athletes is congenital cardiac structural disorders; yet, a significant number of autopsies (44%) cannot identify a cause, these being the autopsy-negative sudden unexplained deaths (AN-SUD) [6,7,12–15].

The current guidelines recommend, for the routine screening for cardiovascular disease in young athletes, the patient's history, a physical examination, and a 12-lead standard ECG, which actually outweighs the first two. Although the cardiac ultrasound could offer significantly more information, especially regarding the congenital anomalies, a lack of evidence restricts its implementation in the standard screening protocols [6,16–22].

There are a few methods that allow the assessment of cardiorespiratory fitness (CRF) in youths which are currently used, such as the 20-m shuttle run test (20mSRT). It is a feasible, reliable, and easy to put in practice method of evaluation, and is currently being discussed to be included in the standardized evaluation protocols [23].

Given the wide spectrum of parameters evaluated (pulmonary, cardiovascular, muscular and oxidative) and the multitude of data and correlations it provides, the cardiopulmonary exercise testing (CPET) is an essential tool in current practice [24]. It is also useful in healthy individuals and in the athletic performance assessment, as maximal oxygen uptake (VO2 max) is directly linked to the exercise capacity [25,26]. Of course, normal parameters vary, as elite aerobic athletes can reach VO2 max values over 80 mL/kg/min, compared to untrained individuals who are in the 30–45 mL/kg/min range [27]. The test can be performed on a treadmill (mostly in the United States) or on the cycle ergometer (mostly in Europe). The cycle ergometer testing has a low associated cardiovascular risk in evaluating young healthy athletes [28,29] (see Table 3).

A particular role of CPET in evaluating athletes is to differentiate between their physiological left-ventricular hypertrophy (LVH) and hypertrophic cardiomyopathy (HCM) [30,31].

**Table 3.** CPET indications (adapted from Löllgen et al. [31]).


Cardiac troponins (cTn) are regulatory components of the cardiac muscle contraction. Cardiac troponin I (cTnI) is of interest due to its phosphorylation sites, which regulate different contractile responses [32,33]. It has been shown that physiological left ventricular

hypertrophy, which is specific to trained persons, causes a reversible increase in Ca2+ dependent force production and in Ca2+-sensitivity in left ventricular (LV) cardiomyocytes. Together with a reduction in cTnI phosphorylation, it is suggestive for an adaptive measure and for the preserved or even increased contractile function, despite the morphology changes [34]. In contrast, untrained persons who are subjected to a sudden physical effort do register increases in cTnI levels, although they are not pathological [35]. Additionally, of interest is the fact that peak levels of hs-cTn are registered between 3 and 4 h after peak physical stress [36].

Myoglobin is a biomarker produced by both the myocardial and skeletal muscle cells. Therefore, its levels rise in cardiac diseases (myocarditis, acute coronary syndromes) as well as muscular stress or strenuous physical effort. It can aid the differential diagnosis of cardiac or muscular damage, whether its levels rise independently or in conjunction with other cardiac biomarkers. Its use in establishing the diagnosis of acute coronary syndrome is low in the absence of other biomarkers' elevation (CK-MB, cTn); however, it is the one whose levels rise earliest in the serum [37–39].

The MB isoenzyme of creatine-kinase (CK-MB) is specific to the cardiac muscle. On its own it cannot predict the cardiovascular risk accurately, unless it is associated with the rise of cTn; thus, its use is still being debated. Its practical utility resides in situations where the estimated glomerular filtration rate (eGFR) is below 15 mL/min/m2, or in recent-onset acute coronary syndromes [40,41].

The N-terminal prohormone of brain natriuretic peptide (NT-proBNP) levels do increase during intense physical exercise, especially in non-trained, amateur athletes. Yet, when not correlated with ECG changes, they are not significant for a cardiovascular pathology [35]. Its release in the blood is more associated with aging and with the effort-generated hypertensive stress on the ventricular and atrial walls [42–44]. Of course, it is essential in diagnosing potential ischemic changes in the myocardium, especially in conjunction with cTn, and with proven benefit in risk stratification in athletes [45–47].

While not being specific markers of myocardial cytolysis, D-dimers levels reflect intravascular fibrinolysis and show higher concentrations in patients with severe atherosclerosis or other causes of coronary stenosis [48]. They have more use in confirming or infirming venous thromboembolism, acute aortic dissection, cardioembolic stroke, or left atrium thrombosis in patients with atrial fibrillation [49]. Additionally, an independent rise in their levels can be specific in persons with implantable cardiac devices [50].

The aim of our study was to assess the relationship between effort-induced cardiac biomarker variations and CPET parameters, and their usefulness in the evaluation of young athletes.

#### **2. Materials and Methods**

#### *2.1. Experimental Approach*

We conducted a complete cardiovascular evaluation of professional football players, with an emphasis on cardiopulmonary exercise testing (CPET) and blood biomarkers (cTnI, myoglobin, CK-MB, NT-proBNP and D-dimers) in order to evaluate and assess the cardiovascular risk. The study was designed following the recommendations for clinical research contained in the Helsinki Declaration of the World Medical Association, and the protocol was approved by both the Ethics Committee of the Clinical Rehabilitation Hospital in Ias,i, Romania, approved on 24 March 2021, and the Ethics Research Committee of the "Grigore T. Popa" University of Medicine and Pharmacy in Ias,i, Romania, nr. 72, approved on 25 April 2021.

#### *2.2. Participants and Protocol*

We evaluated professional football players (n = 19). Football club trainers from the region of Moldova in the counties of Ias,i and Vaslui were informed of the ongoing study and were given our contact data. They themselves informed the registered football players aged between 18 and 20 years, and the first 30 to voluntarily contact us were automatically selected to be evaluated. In order for them to be included, they had to participate actively in regular training during the season of the last year, or at least the last months. Those who had missed part of the last months of training or playing due to injury, or who were in the recovery period after an injury of any sort, were excluded, as well as those with cardio-pulmonary pathological findings during the initial evaluation.

The participants were all male, aged between 18 and 20 years old (mean 18.47 ± 0.841), and fully informed about the procedures, research, and protocols used. They were asked to halt any strenuous training sessions or games a minimum of 24 h before the evaluation, and not to consume any foods or beverages other than water before the initial blood sampling, so as not to interfere with the blood sugar values. Afterwards, they could have a small snack or light meal at least an hour before the CPET. They were admitted through day hospitalization in August and September 2021. Upon admission, they filled out the informed consent forms regarding all the procedures and their inclusion in the study, and underwent rapid COVID-19 antigen testing, which, if negative, would allow them to further proceed with the investigations. They underwent a clinical and paraclinical evaluation with an emphasis on the cardiovascular system, followed by a 12-lead resting ECG, TTE, and CPET. Blood samples for the measurements of cardiac biomarkers values were taken at rest before the procedures and 3 h after finishing the CPET.

#### *2.3. Initial Evaluation*

After the initial blood samples were taken, a complete clinical evaluation was conducted with an emphasis on the cardiovascular system with its four major components (inspection, palpation, percussion and auscultation). The resting blood pressure (BP) and heart rate (HR) values were measured comparatively on the both upper limbs and in the orthostatism using a Rossmax X3 BT automatic blood pressure monitor with a brachial cuff. Height and weight were measured on a SECA digital measuring station.

### *2.4. Resting ECG*

A standard 12-lead resting ECG was performed on a BTL-08 LC device, both in postexpiratory apnea and in deep post-inspiratory apnea, and every morphological change was analyzed to be considered as either normal for an athlete or pathological (see Table 1), and possibly contraindicate further procedures.

#### *2.5. Cardiac Ultrasound*

The standard transthoracic echocardiographic evaluation was performed on a Toshiba Aplio device, prior to the CPET, to assess the cardiac function and to exclude any possible contraindications, using the M-mode, pulse wave Doppler (PWD), continuous wave Doppler (CWD), and color Doppler methods, according to the European Society of Cardiology (ESC) and European Association of Cardiovascular Imaging (EACVI) protocols.

#### *2.6. Cardiopulmonary Exercise Testing*

Functional capacity was assessed by cardiopulmonary exercise testing (CPET) on the BTL CardioPoint software (version 2.32 manufactured by BTL Industries Ltd., Herfordshire, UK) and the BTL-compatible device. We used a progressive maximal symptom-limited CPET protocol on the cycle ergometer, specifically tailored for the athletes: they started on a workload of 15 Watts which was set to increase every 30 s with 12.5 Watts. The duration of the testing was between 10 and 12 min and the recovery period was 10 min.

The most important CPET parameters were: maximal work rate (absolute value, WR (Watt) and percentage of the predicted value, WR% (%)); oxygen uptake with maximal aerobic capacity (absolute value, VO2 max (mL per min) and percentage of the predicted value, VO2 max%); carbon dioxide output (VCO2 (mL per min)); oxygen uptake at the anaerobic threshold (AT) (mL per min); peak value of the respiratory exchange ratio (RER) defined as the ratio between VCO2 and VO2; maximal heart rate (HR (bpm)); O2 pulse as the ratio of VO2 to heart rate, reflecting the amount of O2 extracted per heartbeat; ventilatory efficiency expressing the rise in minute ventilation (VE) relative to VCO2 (VE/VCO2 slope); and heart rate reserve (difference between maximal HR and resting HR, HRR (bpm)). VO2, VCO2, and AT were also expressed as values normalized by body weight (mL per min per km).

Metabolic efficiency was assessed by measuring the increase in VO2 over the rate of increase in work rate (ΔVO2/ΔWR). The slope of this relationship expresses the ability of the muscle to extract O2 during exercise.

Blood pressure was monitored every 2 min using the auscultatory method, while a real-time 12-lead ECG was recorded.

To clinically determine the intensity of the exercise, a subjective rating of the intensity of perceived fatigue was determined by a 6 to 20 Borg scale of perceived exertion.

The test was halted, according to current recommendations, when the subject requested it, upon symptoms or fatigue occurrence, when the blood pressure (BP) measurement exceeded 220 mmHg for the systolic value, or 120 mmHg for the diastolic value, or when suggestive ischemic ECG patterns appeared.

#### *2.7. Cardiac Biomarker Determination*

Peripheral venous blood was collected at rest for the basic laboratory parameters and also to determine the mentioned biomarker values at rest. Three hours after finalizing the CPET, another sample was taken to measure the variations of the biomarker values after the stress test, so as to allow them time to appear and reach certain levels in the blood. All blood sampling was taken from the antecubital veins and in kept dedicated vacutainers (with sodium citrate for D-dimers and lithium heparin for CK-MB, Myo, cTnI, and NTproBNP). Their measurements were performed immediately after, using whole blood on the FIA 8000 device from Getein Biotechnology Co. Ltd. on its dedicated panels: triple tests for CK-MB, myoglobin, and cTnI (CK-MB: measuring range 2.5–80 ng/mL, lower detection limit ≤ 2.5 ng/mL, recovery 96%; myoglobin: measuring range 30–1000 ng/mL, lower detection limit ≤ 30 ng/mL, recovery 95%; cTnI: measuring range 0.5–50 ng/mL, lower detection limit ≤ 0.5 ng/mL, recovery rate 95%, measuring time 15 min), double tests for cTnI and NT-proBNP (cTnI: measuring range 0.5-50 ng/mL, lower detection limit ≤ 0.5 ng/mL, recovery rate 95%; NT-proBNP: measuring range 10–12,000 pg/mL, lower detection limit ≤ 100 ng/mL, recovery 99%, measuring time 18 min) and a single test for D-dimers (D-dimers: measuring range 0.1–10 mg/L, lower detection limit ≤ 0.1 mg/L, recovery rate 99%, measuring time 7 min) were completed. All the assays were stored, according to manufacturer specifications, at a temperature between 4 and 30 degrees Celsius and used before the expiration date was reached and within 1 h since each foil was opened.
