*3.4. CPET*

In 12 out of 19 subjects, the CPET had to be halted due to the recorded systolic BP values of 220 mmHg or higher, while in the remaining 7, the test was stopped due to muscle fatigue. No ECG ischemic patterns were identified, nor any symptoms such as angina were declared. Eleven of them did not reach the 85% heart rate threshold, yet only two had a peak VO2 value below 85% of the predicted. The respiratory exchange ratio (RER) was, in all cases, over 1.00 and only in three cases below 1.0 (see Table 6).

#### *3.5. Cardiac Biomarkers*

cTnI and NT-proBNP levels were below the lower detection limit of the assays in the blood samples in all participants, both at rest and 3 h after the CPET, while seven of the athletes had values below the lower detection limit of the assays for all biomarkers. In four subjects, myoglobin levels increased after 3 h compared to the at-rest values, while in three subjects they decreased. In two participants, CK-MB levels increased post-CPET, while in one athlete they decreased. The D-dimer levels decreased after CPET in five subjects, while in three the values increased. For the statistical analysis, all participants were considered and for those with undetectable values, the lower detection limit value for each biomarker was assigned (see Table 7).



Abbreviations: EDV—end-diastolic volume; ESV—end-systolic volume; SV—stroke volume; EF—ejection fraction; FS—fractional shortening; SI—stroke-volume index; IVSTd—interventricular septum thickness at end-diastole; LVIDd—left ventricular internal dimension at end-diastole; LVPWTd—left ventricular posterior wall thickness at end-diastole; IVSTs—interventricular septum thickness at end-systole; LVIDs—left ventricular internal dimension at end-systole; LVPWTs—left ventricular posterior wall thickness at end-systole; LV MASSd—left ventricular mass at end-diastole; LV MASSd Index—left ventricular mass at end-diastole adjusted to body surface index; LV MASSs—left ventricular mass at end-systole; LV MASSs Index—left ventricular mass at end-systole adjusted to body surface index; E Vel—peak velocity of early diastolic mitral annular motion as determined by pulsed wave Doppler; A Vel—peak velocity of diastolic mitral annular motion as determined by pulsed wave Doppler; E/a—ratio of E to A; A/E—ratio of A to E; DcT—deceleration time MV area; PHT—mitral valve area at pressure half time; PHT—pressure half time; LVOT Diam—left ventricular outflow tract diameter; Ao Diam—aortic annulus diameter; LA Diam—left atrium diameter; LA/Ao—ratio of the left atrial dimension to the aortic annulus dimension.


#### **Table 6.** CPET parameters.

Abbreviations: VO2 max—maximum oxygen uptake; VO2 max body weight—ratio of maximum oxygen uptake to body weight; %VO2 max—percentage of maximum oxygen uptake from the predicted value; VO2@AT oxygen uptake at the anaerobic threshold; VO2@AT body weight—ratio of oxygen uptake at the anaerobic threshold to body weight; RER—respiratory exchange ratio; VE/VCO2—ventilatory equivalent for carbon dioxide; ΔVO2/ΔWR—slope of the relation VO2–power in W; WR max—maximum load; %WR max—percentage of maximum load from the predicted value; O2\_pulse—oxygen pulse; HR max—maximum heart rate; %HR max percentage of maximum heart rate from the predicted value; HR\_rez—heart rate reserve; SBP max—maximum systolic blood pressure; DBP max—maximum diastolic blood pressure.

**Table 7.** Biomarker variation analysis.


#### *3.6. Interrelations between Cardiac Biomarkers and Functional Parameters*

Statistically, in the Mann–Whitney nonparametric tests, CK-MB changes showed significant differences in the heart rate (HR) on the resting ECG (*p* = 0.047), the VO2 max at the CPET (*p* = 0.008), and with the peak load (WR max) on the CPET (*p* = 0.014) between those with and those without changes in CK-MB levels (see Table 8 and Figure 1).


**Table 8.** Significant differences between the groups without changes (No) and with changes (Yes) in CK-MB values.

**Figure 1.** Significant differences between the groups without changes (No) and with changes (Yes) in CK-MB values on resting ECG heart rate (**left**) and on CPET VO2 max (**right**), respectively.

D-dimers showed significant differences within the %WR max on the CPET (*p* = 0.035), between those with and those without changes in D-dimers levels (see Table 9 and Figure 2).

**Table 9.** Significant differences between the groups without changes (No) and with changes (Yes) in D-dimers values.


**Figure 2.** Significant differences between the groups without changes (No) and with changes (Yes) in D-dimers values on CPET %WR max.

Myoglobin showed significant differences between those with and those without changes in myoglobin levels for E/A and A/E ultrasound parameters (*p* = 0.036), VO2 at the anaerobic threshold (VO2 @ AT) on CPET (*p* = 0.045), and with VO2 @ AT/body weight on CPET (*p* = 0.017). Additionally, significant differences were observed with % Watt max on CPET (*p* = 0.045), with % O2 pulse on CPET (p=0.028) and with HR max (*p* = 0.013) and % HR max (*p* = 0.017) on CPET (see Table 10 and Figure 3).

**Table 10.** Significant differences between the groups without changes (No) and with changes (Yes) in myoglobin values.


**Figure 3.** Significant differences between the groups without changes (No) and with changes (Yes) in myoglobin values.

#### **4. Discussion**

Laboratory explorations are gaining more and more ground and sports cardiology represents the new trend in medicine. Therefore, as confirmed by previous studies, their combination is an opportunity for future research [51].

The initial evaluation showed findings consistent with athletes with a normal BMI. In the few cases where BMI was over 25 kg/m2, this was due to the higher muscle mass, as the abdominal circumference was normal. ECG interpretation also showed heart rate and morphology patterns which are considered normal in an athletic population as part of the effort adaptation process (sinus bradycardia, right bundle branch block). The cardiac ultrasound highlighted parameters of increased cardiovascular performance, such as a higher ejection fraction and a mild left ventricular hypertrophy, which, once again, is part of the adaptation to effort process in athletes.

In addition, cardiorespiratory functional evaluations are becoming more used in current practice and screening. As obesity and the sedentary lifestyle are becoming more prevalent, especially among the young population, cardiorespiratory functional evaluations are useful tools for assessing the fitness level. In recent studies, CRF has been proven to correlate negatively with parameters such as BMI and the sedentary lifestyle at younger ages [52].

A study published by Olekšák et al. involved the evaluation of CPET on young Slovenian footballers. They compared the CPET parameters on children and adolescents and concluded that some of them were physiologically higher in athletes and with growing age (VO2 max, Watt max) [53].

The CPET parameters evaluated on the 19 subjects were comparable with those in other studied athlete cohorts. It is interesting to note that some of the registered values, such as a lower VO2 max than predicted, were consistent with the tests which were halted for high BP values. This once again proves the utility of this particular evaluation and shows how physical performance CRF can be limited by an abrupt rise in BP.

Other investigations whose utility has been confirmed in recent studies include biomarker measurements. In a paper published by Mahanty et al., cTn, BNP and hypoxanthine were proven as means of assessing the cardiovascular impact of intense physical activity. Therefore, they are being considered to be implemented in the future as part of screening protocols [54].

A metanalysis published in 2015 concluded that cTnT, hs-cTnT, BNP, NT-proBNP, and D-dimers do suffer serum level changes when a person is performing a high-intensity physical effort, which may interfere with their interpretation in an emergency unit when an acute coronary syndrome, heart failure or pulmonary embolism can be suspected [55].

When investigating young football players, after a full-time football match, cTnI and NT-proBNP levels rose above the baseline and remained elevated even 24 h after the game, yet they never reached pathologically significant values. This rise, compared to our study can be attributed to the higher intensity and duration of the football match (90 min, on average) compared to a CPET [56]. Similarly, blood samples taken from participants in the 2016 Barcelona marathon showed an increase in NT-proBNP levels; however, the intensity of the physical stress and its duration were considerably higher than during a CPET [44].

Another study published in 2021 included individuals that participated in 2018 in the North Sea Race, a 91 km leisure sport mountain bike race. Prior to the race, they performed a CPET. Their cTnI levels were measured before, 3 h, and 24 h after both the race and CPET. The peak values were reached at the 3-h mark, though it should be once again mentioned that, in both cases, the duration and intensity of the physical exercise were higher than in the CPET which we conducted. However, a noticeable inter-individual variation was also observed [57].

There are several mechanisms incriminated for this pattern of variation in biomarker blood levels, such as microvascular ischemia, deficiencies in cardiac metabolism, a systemic inflammatory surge, or even an impaired renal function during intense exercise [58]. This particular dynamic of cTnI concentrations, with an early peak followed by a rapid

normalization within hours (maximum 48–72 h), renders an active myocyte necrosis highly improbable, and rather suggests the above-mentioned secondary mechanisms. In our study, including highly trained athletes, we did not observe any variation of cTnI or NT-proBNP, compared to baseline. These somehow atypical kinetics can be explained by the rather short duration of the CPET, performed by apparently healthy, well-trained individuals, without the deleterious effects of prolonged and exhausting sports that can presumably represent important triggers for the release of cardiac biomarkers. This finding is consistent with the results of Marshall et al., who recently highlighted a similar pattern of troponin fluctuation, with significant variations compared to baseline occurring only in subjects who performed a moderate or intense training regimen. Very interestingly, the same authors noted that a shorter duration of high-intensity exercise induced a more important increase in troponin compared to prolonged, but less intensive, training [59]. Basically, these heterogeneous patterns outline the importance of the duration, intensity, and type of training when assessing cardiac biomarkers. A promising future scenario also assumes the use of novel cardiac biomarkers, such as the soluble suppression of tumorigenesis-2 (sST2) for the early detection of subclinical myocardial injury during sports. Being a marker of increased myocardial strain, fibrosis, and neurohormonal activation, sST2 exhibits a superior prognosis value compared to NT-proBNP or cTnI in patients presenting acute myocardial injury [60,61].

Recent studies have shown that cardiac biomarkers have an important negative predictive value. Thus, low or undetectable hs-cTnI levels can help exclude an inducible myocardial ischemia, in both patients with known coronary artery disease (CAD) and patients without [62–64].

Comparing male and female football players who had CK-MB values measured before, immediately after, and 15 min after a running training session, researchers described a slight increase in CK-MB values immediately post-exercise, most notably in the women's group, yet these values returned to baseline at the 15 min timepoint. The groups comprised both genders and the measurements were performed at different timepoints (immediately after and 15 min after). This study was comparable to ours with regard to the type of sports practiced and the number of participants [65].

Another study measured myoglobin and CK levels immediately after, at the 24 h, 48 h and 72 h timepoints after a high-intensity intermittent running protocol. The results showed a higher increase in myoglobin levels and a more modest one in CK levels, with a return to the baseline values within 24 h for both parameters [66].

On a longer time span, CK and myoglobin levels were measured during a 12-day training period, with blood samples taken prior, at the 6-day mark, and on the 12th day at the end of the training period. CK values peaked on the 6th day, with a drop afterward, while myoglobin peaked on the 12th day [67].

By combining the results of both the cardiac biomarker measurements and of the CPET we can observe how biomarkers are also useful in the assessment of CRF, as they are released into the bloodstream when the cardiovascular stress is at higher values.

The main limitation of our study is represented by the small number of participants, which was not sufficient for more complex statistical analysis tests and could not offer sufficient data for further correlations (see Appendix A).

However, given the low number of publications and existing studies of this design, which combines the complete cardiovascular evaluation of athletes starting from the history, physical examination, 12-lead ECG and cardiac ultrasound and focusing on the combination of the CPET and cardiac biomarker measurements, it is definitely a starting point for further research and future studies. This is also supported by the significant statistical results which were obtained.

Our study offers a more complete approach than other studies, with the combination of CPET and biomarker measurements, and the established correlations so far encourage future research on larger groups, even though the biomarkers which suffered blood level changes (CK-MB, myoglobin, D-dimers) were not specific on their own for coronary diseases [37–41,48]. This association of CPET and biomarkers is also useful to be implemented in cardiac and respiratory rehabilitation evaluations, as shown by a 2021 study conducted by Wang et al., where the improvement of CHF patients' parameters was monitored using these dynamics [68]. Apart from a higher number of subjects, serial measurements of cardiac biomarkers at more timepoints, especially at 12 and 24 h, would offer more indication of their full dynamics in relation to induced physical stress.
