Methods and analysis
Study design
The Master@Heart (Master Athlete’s Heart) study is a multicentre (University Hospitals Leuven, University Hospital Antwerp and Jessa Hospital Hasselt) prospective cohort study, funded by the Fund for Scientific Research, Flanders (T003717N). The study schema is presented in figure 1.
Figure 1Study design and flow chart of the Master@Heart study. From an online screening questionnaire eligible individuals will be sampled and stratified in three groups (lifelong athletes, late-onset athletes, non-athletic controls) of 200 individuals. Baseline evaluation includes an overview of medical history, review of medication and supplements, physical examination, blood sampling, resting 12-lead ECG, two-dimensional and three-dimensional resting echocardiogram (TTE), carotid artery ultrasound, pulse wave velocity for arterial stiffness and non-invasive central blood pressure measurements using a Sphygmocor device, cardiopulmonary exercise testing including 12-lead exercise ECG and maximal oxygen consumption measurement, dual-energy X-ray absorptiometry, CT coronary angiography scan and a 24-hour Holter monitoring. Two hundred and ten subjects, 70 from each group, will undergo cardiac magnetic resonance (CMR) imaging. The latter subjects will also undergo cardiac imaging during exercise, using two-dimensional TTE (Antwerp and Hasselt) or CMR (Leuven). Follow-up consists of a 6-monthly telephone call for clinical events and a 7-day ECG monitoring at 1 year. y, years.
Objectives and hypothesis
The principal aim is to assess the prevalence of mixed, calcified and soft plaques in lifelong endurance athletes compared with endurance athletes who started training later in life and healthy non-athletic controls. The primary hypothesis is that lifelong endurance exercise, more than late-onset training, is associated with a lower prevalence of mixed plaques than non-athletic controls. As secondary aims, the Master@Heart study will assess the association between lifelong endurance training, MF and AF. The hypothesis is that prolonged exposure to endurance exercise is associated with a higher risk of AF and MF. The hypotheses are illustrated in figure 2.
Figure 2Study hypothesis of the Master@Heart study. The primary hypothesis is that lifelong endurance exercise, more than late-onset training, is associated with more calcified plaques and less mixed/and or soft plaques as compared with non-athletic controls. The secondary hypothesis is that prolonged exposure to endurance exercise is associated with a higher risk for atrial fibrillation and myocardial fibrosis.
Study population and eligibility criteria
An online screening questionnaire (www.masteratheart.be; online supplemental appendix) will be used to obtain information on gender, age, weight, length, current and previous smoking behaviour, load and timing of current and prior sports participation, CVRF, medication intake, previous cardiovascular conditions and family history in subjects willing to participate in the Master@Heart study (see online supplemental appendix). The questionnaire was built using REDCap electronic data capture tools hosted at the KU Leuven.47 48 All subjects from this screening cohort will be evaluated for eligibility according to the criteria listed in box 1.
Box 1Eligibility criteria for the Master@Heart study including both inclusion and exclusion criteria.
Inclusion criteria
Men aged 45–70 years.
Athletes engaged in cycling ≥8 hours per week or running ≥6 hours per week or triathlon training (combination of swimming, cycling, running) ≥8 hours per week for at least 6 months prior to baseline.
Non-athletes engaged in ≤3 hours per week of physical activity without prior exposure to regular endurance exercise.
Exclusion criteria
Medical history of cardiovascular disease.
Current or past smoker.
Use of antidiabetic drugs.
Use of statins.
Use of antihypertensive drugs.
Body mass index >27.2 kg/m².
Allergy for iodine contrast agents.
Only male individuals will be eligible for inclusion in the Master@Heart trial because of the higher lifetime risk of coronary heart disease and AF in men relative to women.49 50 Moreover, as the risk of developing coronary heart disease before the age of 40 is low, we opted to include men between 45 and 70 years old.49 To identify an athlete through our online questionnaire, we will use a definition based on the hours per week of endurance training. For runners, we have put forward a cut-off of ≥6 hours per week as a 2016 paper by Dawes and colleagues showed significantly more cardiac remodelling when performing >5 hours of exercise per week instead of <5 hours per week.51 Given the higher intensity of running than cycling, the inclusion criterion for cyclists is ≥8 hours of cycling per week. Since cardiac volumes were similar between sedentary individuals and subjects performing <3 hours per week of exercise, we have chosen a cut-off of <3 hours per week of physical activity to define controls.51
Exclusion criteria
To provide a clear view of the impact of intense endurance exercise on the cardiovascular system, we have decided to minimise the potential impact of CVRF by excluding subjects with known CVRF from the study (box 1).
Smoking, even in minimal doses, significantly increases the risk of coronary heart disease.52 53 The increased risk of atherosclerotic disease persists up to 30 years after smoking cessation.54 Additionally, concurrent or former smoking was a significant limitation in the interpretation of previous studies describing the association between exercise and coronary atherosclerosis.17 18 Therefore, current smoking and any smoking history is an exclusion criterion. A 2010 meta-analysis of nearly 700 000 people reported a twofold increased risk of coronary heart disease associated with diabetes.55 Therefore, the use of any antidiabetic drugs is an exclusion criterion. With regard to lipids, elevated serum cholesterol levels are associated with death from coronary heart disease in middle-aged men during a 12-year follow-up.56 Additionally, statin use has been associated with a higher prevalence of plaque calcification and greater progression of CAC without higher event rates, which has been interpreted as the ability of statins to modulate coronary plaques.57 58 This has been confirmed by serial intravascular ultrasound analysis showing that statin therapy was associated with plaque atheroma regression as well as the progression of plaque calcification.59 Hence, individuals using statins will be excluded from our study. Elevated blood pressure has been related to an increase in cardiovascular events and mortality.60 Individuals using antihypertensive drugs will, therefore, be excluded from the Master@Heart study. Finally, with regard to overweight and obesity, as a body mass index (BMI) between 24.26 and 27.21 did not confer a higher OR for CAC >10 Agatston units, we opted for a BMI cut-off of >27.2 kg/m² as an exclusion criterion.61
From all eligible subjects based on the questionnaire, 600 will be sampled for inclusion. Sampling will be done randomly but stratified by current age (45–53 years, 54–62 years and 63–70 years) as well as age at which endurance training was started (Group 1—lifelong: subdivided in <20 years and 20–30 years, Group 2—late-onset: subdivided in 31–40 years and >40 years, Group 3—healthy non-athletes: NA). This will give equal proportions with regard to current age in all three groups and equal proportions with regard to starting age of endurance exercise in lifelong and late-onset athletes. As the prevalence of high-level endurance athletes is lower in older individuals, we opted for a participant distribution of three out of seven aged 45–53 years, three out of seven aged 54–62 years and one out of seven aged 63–70 years. The sampling and stratification strategy is illustrated in figure 3.
Figure 3Sampling stratification of the Master@Heart study by current age (45–53 years, 54–62 years and 63–70 years) and age at which endurance training was started (lifelong: <20 years and 20–30 years; late-onset: 31–40 years and >40 years; non-athletic controls: N/A). For current age a proportion of 3/7 : 3/7 : 1/7 of individuals aged 45–53 years, 54–62 years and 63–70 years, respectively, was applied.
Study procedures
Baseline evaluation will include an overview of medical history, review of medication and supplements, physical examination, blood sampling for biochemistry and genotyping, resting 12-lead ECG, two-dimensional and three-dimensional resting echocardiogram (TTE), carotid artery ultrasound, pulse wave velocity for arterial stiffness and non-invasive central blood pressure measurements using a Sphygmocor device, cardiopulmonary exercise testing (CPET) including 12-lead exercise ECG and maximal oxygen consumption measurement, dual-energy X-ray absorptiometry, CTCA scan and a 24-hour Holter monitoring in all 600 subjects. CMR imaging, including gadolinium contrast administration, will be performed in 210 randomly selected subjects, 70 from each group. Randomisation for CMR occurs at the initial sampling and stratification with a similar age distribution. This subgroup of study participants will also undergo cardiac imaging during exercise, using two-dimensional TTE (Antwerp and Hasselt) or CMR (Leuven). The sequence of investigations will differ between sites based on logistics.
To gain a broad cross-sectional view on the association between endurance exercise and cardiovascular pathology, subjects will be followed-up for a minimum of 1 year. Follow-up will consist of online tracking of sports activity using TrainingPeaks (Peaksware, Boulder, USA). At 6 months, a telephone call will assess clinical events such as alteration in medication, the onset of AF, coronary interventions or major adverse cardiovascular events. Finally, at 1-year follow-up, a 7-day ECG-monitoring will identify AF and other arrhythmias.
A clinical report covering all relevant findings will be available to the patient and his general practitioner. For research purposes, all measurements and recorded events will be pseudonymised and stored in the REDCap database.
Physical examination and blood samples
The information derived from the physical examination and blood sampling, such as systolic and diastolic blood pressure, total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, will be used to assess the individual cardiovascular risk score.
12-lead ECG
A resting 12-lead ECG will be recorded and interpreted following the Seattle Criteria by a cardiologist with experience in sports medicine.62
Sphygmocor
Carotid–femoral pulse wave velocity is a well-established marker of arterial stiffness and is linked to total cardiovascular risk.63 Using a SphygmoCor XCEL device (SphygmoCor device, AtCor Medical, Sydney, Australia), the central blood pressure, the central arterial pressure waveform, central aortic pressures and pulse wave velocities will be measured, allowing assessment of arterial stiffness. Pulse wave velocity is determined by simultaneous measurement of the carotid and femoral pulse by tonometry and volumetric displacement. The transit time between the feet of the two waves is calculated. Distances are measured from the suprasternal notch to the top of the thigh cuff, the site of carotid tonometry and the site where the femoral artery could be applanated by tonometry.64
Dual-X-ray absorptiometry
Subjects will undergo a dual-energy X-ray absorptiometry scan (Discovery W, Hologic, Bedford, Massachusetts, USA - GE Lunar Prodigy Advance, GE Healthcare, Horten, Norway) to measure lean mass, fat mass, bone mineral content and bone mineral density of the whole body, trunk, legs and arms.
Cardiopulmonary exercise testing
Peak oxygen consumption (peak VO2) will be determined using a continuous bicycle stress test. After a 5 min warm-up, resistance will gradually increase by 30W every minute from an initial load of 60W in cyclist and 30W in runners and controls until exhaustion. ECG monitoring during the test will ensure analysis of heart rate, arrhythmias and repolarisation abnormalities. During the exercise test’s final stages, the respiratory gas exchange will be analysed using a breath-by-breath open-circuit spirometry system. Peak VO2 will be determined as the highest 30 s average oxygen consumption. The first and second ventilatory threshold will be determined from respiratory gas analysis parameters.
Two-dimensional and three-dimensional transthoracic echocardiography
Two-dimensional and three-dimensional TTE will be performed using a Vivid E9 or E95 ultrasound system (GE Healthcare, Horten, Norway) with an active matrix single-crystal phased array transducer (GE M5Sc-D probe, GE Healthcare, Horten, Norway) and 1.5–4MHz matrix-array transducer (GE 4Vc-D Matrix 4D cardiac probe, GE Healthcare, Horten, Norway). Cardiac morphology will be assessed, including end-diastolic volume, end-systolic volume, rendering ejection fraction (EF) for both ventricles, as well as right and left atrial volumes. The diastolic function will be assessed using established Doppler and tissue-Doppler parameters such as the E wave velocity, the A wave velocity, the E/A ratio, septal, lateral and averaged E’, E/E’, tricuspid regurgitation flow velocity and the S-D-A waves at the pulmonary veins. An in-depth analysis of the intrinsic myocardial function will be performed by strain analyses. RV and LV strain and strain rate will be assessed as systolic function measures. RV and LV early and late diastolic strain rate will be assessed for diastolic function. Time-to-peak shortening in all 18 segments of the LV and the RV free wall will be measured to assess for differences in timing and mechanical dispersion. Atrial strain analysis will be performed to assess the reservoir, conduit and contraction function of both atria. All measurements will be made following international guidelines.65 66
Carotid artery ultrasound
The presence of atherosclerotic plaques at the common carotid artery and internal carotid artery will be evaluated using a ProSound Aloka Alpha 6 (Aloka Holding Europe AG, Zug, Switzerland), a Philips Epiq5 (Philips Medical Systems, Bothell, Washington, USA) or a Vivid E9 (GE Healthcare, Horten, Norway) ultrasound system with a UST-5413 (Aloka Holding Europe AG, Zug, Switzerland), L12-3 (Philips Medical Systems, Bothell, Washington, USA) or Vivid E9 (GE Healthcare, Horten, Norway) or a 9L (GE Healthcare, Horten, Norway) transducer. Pulse wave Doppler measurement will be used to assess the degree of stenosis when plaques are present. To further assess peripheral atherosclerotic burden, the intima-media thickness will be measured as the distance between the hyperechogenic blood-intima line and the hypoechogenic media-adventitia line at the distal part (1 cm proximal to the bulb) of the common carotid artery and the proximal part (1 cm distal to the bulb) of the internal carotid artery. All measurements will be made following international guidelines.67 68
CTCA
CTCA will be acquired using a 128-slice dual-source CT scanner (Siemens Somatom Force—Siemens Healthineers, Forchheim, Germany) or a 256-slice CT scanner (GE Revolution—GE Healthcare, Milwaukee, Wisconsin) or a 320-slice CT scanner (Aquilion ONE ViSION—Canon Medical Systems, Otawara, Japan). To achieve a target heart rate of <65 beats per minute, the beta-blocker esmolol will be used intravenously when necessary. The choice for esmolol instead of the more conventionally used beta-blockers (ie, metoprolol) was based on esmolol’s short half-life, which will prevent interference with other tests (ECG, CPET, TTE, CMR and Holter). All subjects will receive 0.4 mg sublingual nitroglycerine 2 min before scanning. First, a non-enhanced ECG-synchronised scan will be taken for the quantification of coronary calcium rendering the CAC score. Second, an ECG-triggered CTCA will be acquired after the intravenous injection of iodinated contrast medium in an antecubital vein followed by a saline chaser. All coronary atherosclerotic lesions will be analysed and interpreted following the 2016 Society of Cardiovascular Computed Tomography (SCCT) guidelines on syngo.via (Siemens Healthineers, Forchheim, Germany) or GE Advanced Workstation (GE Healthcare, Milwaukee, Wisconsin) software with regard to their stenosis grade by the Coronary Artery Disease Reporting and Data System (CAD-RADS), composition (calcified, mixed, soft) as well as for their vulnerability (positive remodelling, low-attenuation plaque, spotty calcification and the napkin-ring sign). A density of >130 HU defines calcified areas. Calcified plaques are entirely composed of calcified areas. Mixed plaques are composed of both calcified and non-calcified areas. Soft plaques are entirely composed of non-calcified areas. The use of the syngo.via Frontier Coronary Plaque Analysis software (Siemens Healthineers, Forchheim, Germany) will further assess luminal and plaque volumes.69–71
Cardiac MRI
In 210 randomly selected participants (70 per group, matched for age), CMR will be performed using a 1.5T MRI scanner (Magnetom Aera 1.5T—Siemens Healthineers, Erlangen, Germany; Ingenia, Achieva or Ambition 1.5T—Philips Medical Systems, Best, The Netherlands), a dedicated cardiac coil and electrocardiographic gating. Steady-state free precision short-axis cine imaging (8 mm slice thickness without gaps) will be obtained to analyse cardiac mass, function and volumes. Also, native and post-contrast T1 mapping will be performed using the modified look-locker inversion recovery sequence to calculate ECV. MF will also be evaluated using delayed enhancement of breath-hold phase-sensitive inversion recovery sequences 10 min after administering gadolinium-diethylenetriamine pentaacetic acid. Analysis of all CMR data will be performed in a central core laboratory. Assessment of cardiac volumes and mass will be performed using CVI42 (Circle Cardiovascular Imaging, Calgary, Canada). IntelliSpace Portal (Philips Medical Systems, Eindhoven, The Netherlands) will be used for T1 and ECV mapping, whereas suiteHEART (NeoSoft, Pewaukee, USA) is used for strain analysis (feature tracking). Our validated robust non-rigid motion correction will be used for accurate T1 measurements and ECV calculations.72 T1-mapping will also provide an estimate of myocardial cellular mass as a feature of athletic remodelling.73 In University Hospitals, Leuven CMR imaging will also be performed during exercise. Our research group has previously demonstrated the feasibility, reliability and clinical utility of CMR to quantify cardiac volumes and function during exercise.74–76 The assessment of myocardial function during exercise will allow us to investigate whether the duration and intensity of long-term endurance exercise affect LV and RV functional reserve.
Exercise stress echocardiography
In the University Hospital Antwerp and Jessa Hospital Hasselt, exercise stress echocardiography (Vivid E95 ultrasound system—GE Healthcare, Horten, Norway) will be performed instead of exercise CMR in the subjects having undergone CMR at rest. Measurements will be performed at rest and during several exercise stages depending on heart rate and respiratory gas analysis parameters. The following measurements and derived calculations will be collected: left ventricular ejection fraction (LVEF), RVEF, RV fractional area change, biventricular systolic and diastolic strain parameters, Doppler and tissue Doppler parameters to assess cardiac output, diastolic function and pulmonary artery systolic pressure. Using stress echocardiography, we will assess how prolonged high-intensity endurance training impacts pulmonary vascular resistance and diastolic function during exercise. All measurements will be made following international guidelines.77
Holter analysis
For the 24-hour Holter ECG monitoring, a Spiderview Holter device (ELA Medical, Paris, France) will be attached to BlueSensor VL ECG electrodes (Ambu, Penang, Malaysia). The ECG recordings will be analysed offline using SyneScope software (ELA Medical, Paris, France) to determine heart rate boundaries and to evaluate the occurrence of arrhythmias. Bradycardia is defined as a heart rate slower than 50 beats per min. A cardiac pause is defined as an interruption in the ventricular rate >2 s and non-sustained ventricular tachycardia as three or more consecutive ventricular beats (origin below atrioventricular node) with an RR interval <600 ms (ie, >100 beats per min) and lasting <30 s.
Seven-day ECG monitoring
For the 7-day ECG monitoring, a RootiRx (Rooti Labs, Taipei, Taiwan) will be used. RootiRx is an ECG patch monitoring device consisting of an integrated sensor system, a microelectronic board with memory storage and an internal rechargeable battery. RootiRx allows for continuous ECG monitoring for up to 7 days in 250 Hz frequencies with 24-bit high resolution. Recorded data is analysed by Rooti Labs developed algorithms and creates a report which is reviewed and edited by physicians and finally sent back to the referring physician. The final report includes the number of recording days, the amount of recorded beats, the average heart rate (overall, day and night), the maximum and minimum heart rate, the amount of cardiac pauses defined as an interruption in the ventricular rate >2 s. AF burden is reported as an amount of AF events, time in AF and percentage time in AF. Reported atrial events include atrial ectopic beats and supraventricular tachycardia, and ventricular events include ventricular ectopic beats, doublets, triplets, bigeminy, trigeminy and ventricular tachycardia. The performance of RootiRx has been validated against standard 24-hour Holter monitoring in healthy individuals and patients with arrhythmias.78
Endpoints
The Master@Heart study’s primary endpoint is the difference in the prevalence of mixed plaques in lifelong endurance athletes, late-onset endurance athletes and non-athletic controls. The main secondary endpoints are (1) prevalence of AF on 12-lead ECG, a 24-hour Holter monitoring or a 7-day ECG-monitoring, (2) the presence and quantification of MF as assessed by LGE imaging (% of LV mass) and T1-mapping (% of ECV) and (3) total CAC scores and the presence of >50% stenosis in proximal coronary segments. Tertiary endpoints include: (1) prevalence of ventricular ectopic beats, non-sustained and sustained ventricular tachycardia, a 24-hour Holter monitoring or a 7-day ECG monitoring; (2) LV and RV systolic function at rest and during exercise; (3) LV diastolic function by two-dimensional echocardiography including speckle tracking imaging; (4) bi-atrial function by two-dimensional echocardiography including speckle tracking imaging; (5) filling pressures and pulmonary artery pressures during exercise assessed by exercise echocardiography; (6) arterial stiffness by Sphygmocor; and (7) carotid intima media thickness as assessed by carotid artery ultrasound.