Influence of a 7‐day Transalpine Trail Run on cardiac biomarkers and myocardial function

Intense physical exercise is known to increase cardiac biomarkers; however, it is unclear, whether this phenomenon is physiological, or if it indicates myocardial tissue injury. The aim of our study was to investigate the effects of seven consecutive days of excessive endurance exercise on continuous assessment of cardiac biomarkers, function, and tissue injury. During a 7‐day trail‐running competition (Transalpine Run, distance 267.4 km, altitude ascent/descent 15556/14450 m), daily blood samples were obtained for cardiac biomarkers (hs‐TnT, NT‐proBNP, and suppression of tumorigenicity‐2 protein (ST2)) at baseline, after each stage and 24–48 h post‐race. In addition, echocardiography was performed every second day, cardiac magnetic resonance imaging (CMR) before (n = 7) and after (n = 16) the race. Twelve (eight males) out of 17 healthy athletes finished all seven stages (average total finish time: 43 ± 8 h). Only NT‐proBNP increased significantly (3.6‐fold, p = 0.009) during the first stage and continued to increase during the race. Hs‐TnT revealed an incremental trend during the first day (2.7‐fold increase, p = 0.098) and remained within the pathological range throughout the race. ST2 levels did not change during the race. All cardiac biomarkers completely returned to physiological levels post‐race. NT‐proBNP kinetics correlated significantly with mild transient reductions in right ventricular function (assessed by TAPSE, tricuspid annular plane systolic function; r = −0.716; p = 0.014). No significant echocardiographic changes in LV dimensions, LV function, or relevant alterations in CMR were observed post‐race. In summary, this study shows that prolonged, repetitive, high‐volume exercise induced a transient, significant increase in NT‐proBNP associated with right ventricular dysfunction without corresponding left ventricular functional or structural impairment.

and 24-48 h post-race.In addition, echocardiography was performed every second day, cardiac magnetic resonance imaging (CMR) before (n = 7) and after (n = 16) the race.Twelve (eight males) out of 17 healthy athletes finished all seven stages (average total finish time: 43 ± 8 h).Only NT-proBNP increased significantly (3.6fold, p = 0.009) during the first stage and continued to increase during the race.
Hs-TnT revealed an incremental trend during the first day (2.7-fold increase, p = 0.098) and remained within the pathological range throughout the race.ST2 levels did not change during the race.All cardiac biomarkers completely returned to physiological levels post-race.NT-proBNP kinetics correlated significantly with mild transient reductions in right ventricular function (assessed by TAPSE,

| INTRODUCTION
In sports cardiology, there is an ongoing debate whether prolonged and strenuous exercise like a marathon or ultramarathon may induce myocardial dysfunction or even injury.In light of this debate, an upper-dose exercise limit, beyond which adverse effects of exercise may outweigh its benefits is discussed. 1,2It is known that strenuous endurance exercise, like running a marathon, induces significant elevations of cardiac biomarkers in healthy individuals.However, these changes are transient, and concentrations return to normal levels within 24 h. 3,4These increases are dependent on physical fitness level, exercise intensity, exercise volume of the corresponding exercise activity, and renal function. 3,5Exercise-induced troponin increases seem to be a physiological phenomenon when rapid normalization is observed post-race-even if it is related to intense and vigorous exercise, a pattern distinct from the long-term changes which occur during myocardial infarction with prolonged elevations. 6,7he majority of studies examining cardiac changes in response to exercise describe functional and structural myocardial alterations due to strenuous exercise, classified as "cardiac fatigue." 85][16] Most of the described effects are rapidly reversible. 3,8,9,12,16he role of repetitive strenuous exercise on cardiac biomarkers and myocardial function has scarcely been investigated.Therefore, the aim of our study was to investigate the effects of the Transalpine Run (TAR), a multistage competitive race across the Alps consisting of seven consecutive days of mountainous trail running in healthy athletes of both genders.We hypothesized that the TAR would pose a significant strain on the peripheral musculature as well as myocardium.We particularly aimed to determine whether the consecutive nature of such a race would induce a progressive impairment of cardiac function as assessed by post-race echocardiography or reveal myocardial injury assessed by post-race cardiac magnetic resonance imaging (CMR).

| Study design
We performed an observational, longitudinal pilot study during the TAR, a seven-stage trail-running competition across the Alps through four different countries (Germany, Austria, Switzerland, and Italy).The race consists of a total of 267.4 km (15 556 m uphill; 14 450 m downhill) run on seven consecutive days (average distance of 38.2 km/day; 2222 vertical meters of uphill/day (range: 24.2-46.5 km/stage, 1692-2930 vertical meters/stage)).Detailed information on the distance and vertical meters (uphill and downhill) per day/stage is shown in Table 1.After each of the seven stages, blood was drawn for laboratory analysis.Echocardiography was performed directly after every second stage, and a cardiac MRI (CMR) was performed on the first two post-race days.Participants had been thoroughly examined 14 days prior to competition, including blood analysis, echocardiography, exercise test, and in n = 7 a CMR.All of these participants had signed an informed consent form beforehand to participate in the study.The study protocol had been approved by the local ethics committee (373/17S) and was conducted in accordance with the Declaration of Helsinki.

| Subjects
Two months prior to the race, all pre-registered athletes for the TAR had been contacted by the TAR organizing committee (Plan B event company GmbH) by email to gauge interest in the study.To be eligible for enrollment, interested subjects had to fulfill the following inclusion criteria: intention to participate in the TAR 2017, healthy tricuspid annular plane systolic function; r = −0.716;p = 0.014).No significant echocardiographic changes in LV dimensions, LV function, or relevant alterations in CMR were observed post-race.In summary, this study shows that prolonged, repetitive, high-volume exercise induced a transient, significant increase in NT-proBNP associated with right ventricular dysfunction without corresponding left ventricular functional or structural impairment.

K E Y W O R D S
cardiomyopathy, CMR, exercise, NT-proBNP, running, troponin medical condition, age ≥18 years, and ability to provide written informed consent.Exclusion criteria were history of or current cardiac disease, liver or kidney disease, neoplasia, orthopedic limitations which would restrain the participant from running, acute or chronic infections, diabetes mellitus, drug, medication or alcohol addiction, and mental limitations leading to be incapable of acting lawfully.

| Clinical assessments
Baseline assessment (2 weeks before the race, T0) included medical and training history (weekly running distance and running experience/total training age), physical examination, anthropometry, collection of blood samples, 12-lead electrocardiogram (ECG), echocardiography, cardiopulmonary exercise testing (CPET, Cortex MetaLyzer 3B) according to current standards, 17 and CMR 18 in n = 7 athletes.The other 10 participants refrained from prestudy CMR analysis.
During the race, blood samples were collected after each stage (Day 1 to 7; T1-T7; <15 min after finish), centrifuged, aliquoted, and frozen for later analysis.In addition, echocardiography was performed directly after the third, fifth, and seventh stages (T3, T5, and T7).Within 24-48 h after finishing the TAR (T8), additional blood samples were collected and a CMR was performed at the Department of Interventional and Diagnostic Radiology, Klinikum rechts der Isar, Munich, Germany.
2D-Echocardiography (Philips EPIQ 7G at T0; Transducer: Philips X5-1; Philips Portable CX50 at T1-T7) was performed by the same investigator, in accordance with the American Society of Echocardiography (ASE) and European Association of Cardiovascular Imaging (EACVI) guidelines. 19,20CMR was performed according to recommendations of the Society of Cardiovascular Magnetic Resonance 18 (3.0Tesla Philips Ingenia clinical dual-source RF transmission MR system; Philips, Germany) using a five-element cardiac phased-array coil with an omega HP gradient system.Thereby, standardized apical, mid-ventricular, and basal short-axes as well as two-and four-chamber views were performed.In order to detect signs of myocardial edema or fibrosis, T1 and T2 mapping as well as T2-weighted (T2w) fat saturation were assessed.Additionally, pericardial effusion was evaluated.Analyses of T1 and T2 mapping were performed by applying a 16-segment model according to the American Heart Association. 19

| Statistical analysis
Statistical analyses were performed using SPSS Statistics 25 (SPSS Inc., Chicago, IL).All parameters were tested for normality using the Shapiro-Wilk test.Continuous variables were reported as mean ± SD if normally distributed, or as median (interquartile range IQR = 25th-75th percentile) if not normally distributed.Repeated measures ANOVA was used to compare normally distributed variables followed by the post-hoc Bonferroni test.In case of non-normally skewed distribution, transformation by natural logarithm was applied before parametric data analyses.For testing relationships between two parameters, Pearson's correlation coefficient was calculated.Statistical significance was assumed at p < 0.05.

| Subjects characteristics
Seventeen (71% male; age 37 ± 8 years) healthy ultraendurance runners out of the initially screened 18 participants (one excluded because of arterial hypertension) were included in the TAR-Study.Twelve athletes (66% male) finished all seven stages, for all of which both laboratory and echocardiographic results could be obtained.Four athletes were not able to finish all stages because of muscular fatigue/injury, one athlete because of a common
CK and GPT, both parameters of muscular strain, increased significantly 14.3-fold (p < 0.001) and 3.9-fold (p = 0.002), respectively, from T0 to T4.These values were still clearly above the upper reference limit even 24 h after the race.
No clinical cardiac signs or symptoms were reported during or after the race.All participants complained of moderate-to-severe muscular fatigue and muscle soreness.

| Echocardiography
All LV parameters, including LVEF, remained constant during the multistage race (Table 4, Figure 3), and none of the investigated values were out of normal range. 19V data are shown in Figure 3A-C.When compared to baseline, all measures of systolic RV function, assessed by TAPSE and FAC, were reduced during the race: TAPSE decreased significantly at T3 (p = 0.014) and T5 (p = 0.016) when compared to baseline values, but remained within normal range throughout the race.Additionally, FAC declined, albeit insignificantly (from 43 ± 13.3 at baseline to 31.0 ± 9.9 at stage 3 (p = 0.272)), and persisted at the same level until the end of the race (T7).For diastolic RV function, a transient trend from T0 to T3 (p = 0.153) with increased values compared with baseline values was observed.
Multiple regression analyses revealed a significant association between the increment in hs-TnT concentration Mean ± standard deviation

95% confidence range
Age   (hs-TnT measured at baseline vs. after T7) and low exercise performance, for example, running speed (Figure 4A, r = −0.61;p = 0.037).In further bivariate correlation analyses, we found a significant relationship (r = −0.72,p = 0.013) between increment in NT-proBNP during the race (NT-proBNP measured at baseline vs. after T7) and change in TAPSE (TAPSE quantified at baseline vs. after T7) (Figure 4B).No significant correlations were observed between finish time and changes in NT-proBNP or TAPSE (all p-values >0.05).

| Cardiac magnetic resonance imaging
Cardiac magnetic resonance imaging (CMR) could be performed in n = 7 before the race and n = 16 afterward (n = 1 received no CMR because of logistical reasons).All finalists (n = 12) received a CMR after the race (n = 4 drop-outs).In five, we obtained a CMR before and after the race.In one woman a pericardial effusion of a maximum of 5 mm was present before the race, however, she revealed no clinical symptoms or signs of cardiac dysfunction and participated in the run after a thorough cardiological examination and informed consent under these circumstances.The pericardial effusion did not change during the race.We observed no signs of myocardial edema in all post-CMR images (Table 5).There was no difference in CMR images between pre-and post-race in the subgroup of n = 5.

| DISCUSSION
Previous studies have evaluated cardiac function following endurance exercise such as marathons, ironman triathlons, or mountain ultramarathons. 3,8,9,12,16,21Here, we report on alterations in cardiac biomarkers as well as cardiac function and structure during a multistage exercise challenge with extremely high peripheral muscular strain, with short recovery periods between stages.As a main finding, the TAR induced a significant peripheral muscular strain and injury seen by 14.4-fold increases in CK without significant increases in hs-TNT, but a significant increase in NT-proBNP throughout the race.The latter correlated to right ventricular function impairment with immediate normalization within 24-48 h post-race.CMR after the race also revealed no evidence of myocardial edema or structural injury.Thus, our results provide no evidence for acute cardiac injury by an extreme excessive endurance competition in healthy athletes of both genders.
In accordance with previous studies, 3,4,14,22 a mild-tomoderate elevation of all cardiac biomarkers was observed at the beginning of the race (e.g., hs-TnT concentration increased 2.8-fold from baseline to end of stage 1).Increases in these biomarkers during marathon races have previously been proven significantly higher than during the TAR, with hs-TnT levels increasing by 10.8-fold immediately after the marathon race (mean finish time 3:47 ± 0:26 h, pace of 5.4 min/km). 3Running distance was similar, but cardiopulmonary exercise intensity during TAR was clearly lower (9.6 min/km) than in a single-day marathon race.Nonetheless, we also found a significant positive correlation between running pace and increment of hs-TnT (Figure 4A).Thus, the fastest finisher of TAR (pace: 6.4 min/km) revealed the highest hs-TnT levels of all participants.These findings overall indicate that exercise intensity may be an important factor for exerciseinduced hs-TnT increases.Contrary to our expectations, troponin did not accumulate, but remained at a similar level over the duration of the entire race.However, values were within the pathological range throughout the race.results have previously been reported during a multistage marathon of 4486 km, which showed a moderate troponin elevation (2.5-fold) during the first measurement after 1000 km, with no further significant increase thereafter. 21In our study, all participants showed rapid normalization of hs-TnT levels within 24-48 h after TAR.The association between prolonged elevation of postexercise troponin concentrations and the presence of coronary artery disease (CAD) has been assessed in the Norwegian NEEDED trial, in which 133 subjects (mean age 44 ± 11 years) underwent serial troponin measurements after a 91 km mountain-bike race.Additionally, cardiac computed tomography angiography (CCTA) was performed to search for CAD.Data revealed that markedly prolonged elevated troponin levels after the race were indicative of occult CAD, 23 whereas rapid normalization of exercise-induced hs-TnT was not related to CAD and therefore interpreted as a physiological process.
T A B L E 4 Echocardiographic data at baseline and twice during the Transalpine Run (TAR) (T3 and T5) and post-run (T8).

Echocardiography
Baseline T3 T5 T8 LVEDD [cm] 5.2 ± 0.5 4.9 ± 0.6 4.9 ± 0.4 5.0 ± 0. However, NT-proBNP showed different kinetics compared with hs-TnT.After a significant increase during the first race day, it remained at a similar level until Day 4 and showed a second peak toward the end of the week.NT-proBNP induces a reduction in cardiac wall stress by natriuresis, vasodilation, and sympathoinhibitory action. 24Consequently, it has been hypothesized that an increase in NT-proBNP may indicate cytoprotective and growth-regulating effects and represents a physiological cardioprotective response. 24,25charhag et al. reported elevated NT-proBNP levels in 77% of participants in endurance competitions, for example, marathon, 100 km run, and mountain-bike marathon. 4100 km runners had the highest NT-proBNP concentrations after exercise and directly correlated with exercise time and cardiac wall stress. 4In our study, the highest NT-proBNP levels were also observed in the youngest athlete with the least training experience and lowest exercise training volume per week, a finding similarly reported in previous studies. 26owever, NT-pro-BNP kinetics were correlated significantly with reductions in systolic RV function assessed by TAPSE in echocardiography (Figure 4A).TAPSE decreased significantly during the first 3 days of the run, and plateaued thereafter.Similar findings were described by La Gerche et al., who studied 40 athletes competing in endurance events with a duration of three to 11 h. 27A significant correlation between changes in RV function and post-race NT-proBNP levels was observed, and was most closely correlated to race duration.Thus, NT-proBNP may provide a predictor of both acute RV load and subsequent fatigue during sustained load. 27Despite the exercise-induced depression of systolic RV function, there is strong evidence suggesting this as a transient, fully reversible phenomenon in healthy athletes. 28In our study, diastolic RV function tended to decrease during the first 3 days, but this did not

(B) (A)
significance, and almost returned to the baseline level thereafter.It seems that diastolic RV dysfunction is primarily related to intensity rather than duration.After marathon running, a more persistent diastolic RV dysfunction was observed, 29 whereas an Ironman triathlon (3.9 km swim, 180.2 km bike ride, and 42.2 km run) led to a transient reduction in diastolic RV function with normalization during the recovery period. 16Our study also revealed only transient RV diastolic dysfunction in a race of the slow running pace of 9.6 ± 1.8 min/km during the TAR, which is equivalent to brisk walking.
These results are consistent with the majority of recent studies, which showed either a mild impairment of LV function immediately post-exercise, [9][10][11] or no changes in LV measurements at all. 12,13,27Our findings add to these data, showing no significant changes in CMR directly after the race in large sample of athletes of both genders.Particularly, no clinically relevant alterations, especially no signs of myocardial edema or pericardial effusion, were observed in any of the participants, a clear sign that elevations of cardiac biomarkers, when transient, do not resemble myocardial cell injury.This is in accordance with findings of the Trans Europe Foot Race 2009, an ultramarathon race held over 64 consecutive days and 4486 km, which also revealed no evidence of myocardial injury. 21

| CONCLUSION
The Transalpine Run induced a transient increase in NT-proBNP related to minor impairment of right ventricular systolic function, but rapid normalization after the race.Moreover, our results from CMR in a subgroup of athletes provide no evidence for persisting myocardial injury caused by such an extreme excessive endurance competition with seven consecutive days of running up-and downhill and only short recovery periods.

| LIMITATIONS
Several limitations of our study should be emphasized: A very important limitation is the lack of the assessment of further RV parameters as the right ventricle is the heart chamber that is most affected by exercise.Furthermore, speckle tracking has not been performed.It would have been beneficial including strain analyses of both ventricles.Due to logistical reasons, CMR could only be performed in a small subgroup.

| PERSPECTIVES
Future research will be important to examine altered cardiac and inflammatory biomarker levels with regard to myocardial injury during and after excessive endurance training.Although this study is certainly unique in the way that we investigated CMR after this repetitive exercise challenge, investigating CMR at baseline in all athletes also including late gadolinium enhancement analysis would have been favorable.Moreover, blood analysis could have been extended to measurements before each stage.Findings via imaging will be of interest to coach endurance athletes concerning putatively induced myocardial dysfunction.Athlete with pericardial effusion at T0 was included in the analysis.

F I G U R E 1
Flow chart of the participants of the study.

T A B L E 2
Baseline data and training characteristics in 17 participants (12 male).T A B L E 3 Blood values at baseline and after each stage of TAR (Day 1-7, T1-T7) and post-race (Day 8, T8).

T A B L E 5
Cardiac MRI data at baseline and after the race (T8).

a
Against baseline (T8 = 24-48 h after the TAR; T2w: T2-weighted fat saturation).N = 5 represent corresponding values at baseline and at T8, N = 12 represent all finishers at T8. b Distance and vertical meters (uphill, downhill) per day/stage (T1-T7) of the Transalpine Run (TAR).T1-T7 are the stages during the 7-day Transalpine Run.cold.A flow chart of participants is shown in Figure1.Baseline data and training characteristics of participants are summarized in Table2.The average total finish time was 42.8 ± 7.8 h, with a mean pace of 9.6 ± 1.8 min/km.The fastest runner needed a total of 28.6 h to complete the seven stages.
T A B L E 1Note: Variables are reported as mean ± SD when normally distributed, p-values represent statistical differences in previous examinations. Note: Variables are reported as median (interquartile ranges) when not normally distributed. Note: