Multi-level Molecular Profiling of Peak Performance in Endurance Sports

Study Overview
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Brief Summary
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Physical activity triggers complex molecular responses, including changes in immune-, stress-, and metabolic pathways. For example, autophagy is essential for energy and cellular homeostasis through protein catabolism, and dysregulation results in compromised proteostasis, reduced exercise performance, and excessive secretion of signaling molecules and inflammatory proteins. However, previous research has been limited by the extend of molecules measured and biological processes covered. A better understanding of these processes through multi-omic analysis can improve knowledge of molecular changes in response to exercise. The main purpose of the investigators study is to analyze the effects of acute exercise in correlation to autophagy and other signaling cascades. Specifically, the investigators plan to perform multi-level molecular profiling in a cohort of healthy male elite cyclists and male and female recreational athletes, before, during, and after a bicycle ergometer test. The results will be compared to a control cohort without intervention.

Detailed Description
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This is a non-randomized controlled trial performed at the Paracelsus Medical University, Salzburg, Austria. The study will recruit 80 healthy men and women. Subjects who meet the inclusion criteria will be allocated to four arms (n = 20 in all groups): 1. elite cyclists, 2. male recreational athletes, 3. female recreational athletes, 4. male control group. After overnight fasting and medical check-up, groups 1-3 will undergo a bicycle ergometer-based exercise protocol designed to span low (aerobic) to severe (anaerobic) domains of exercise. The protocol consists of a 15 min aerobic warm-up phase followed by a ramp-bicycle ergometer protocol. During exercise, performance-relevant data will be continuously monitored. Venous blood specimens will be collected before exercise (baseline), at the end of the warm-up as well as 2 min, 10 min, and 30 min in recovery.

Official Title
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A Pilot-study on Multi-level Molecular Profiling of Peak Performance in Endurance Sports

Conditions
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Exercise, Exercise Test, Physical Fitness, Healthy

Intervention / Treatment
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* Diagnostic Test: Performance testing


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: All arms: ability to give written informed consent Arms 1- 3: inconspicuous medical examination (medical history, resting ECG, echocardiography) Elite athletes (arm1, cyclists): maximum oxygen uptake > 65 ml/kg/KG Elite athletes (arm1, cyclists): participation in cycling competitions on a regular basis Recreational athletes (arms 2 and 3): maximum oxygen uptake < 65 ml/kg/KG for male subjects and < 55 ml/kg/KG for female subjects Exclusion Criteria: All arms: unable to communicate adequately by language All arms: regular use of prescription drugs other than thyroxine or antihistamines All arms: alcohol consumption as equivalent doses averaging more than 40 g of pure alcohol per day All arms: use of illicit drugs All arms: known diseases of the cardiovascular system All arms: arterial hypertension over 160/90 mmHg at rest All arms: known pulmonary diseases, especially bronchial asthma All arms: surgery less than 4-6 months ago. All arms: abnormalities in the medical examination (medical history, resting ECG, echocardiography) Arms 1-3: orthopaedic diseases that preclude maximum exercise on a bicycle ergometer Elite athletes (arm1, cyclists): maximum oxygen uptake < 65 ml/kg/KG Recreational athletes (arms 2 and 3): maximum oxygen uptake > 65 ml/kg/KG for male subjects and > 55 ml/kg/KG for female subjects Arm 3: positive urine ß-HCG

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 40 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: 3 groups undergoing intervention, 1 control group
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Elite cyclists, male<br>Bicycle ergometer-based exercise testing. 15 min aerobic warm-up phase (2 W/kg) followed by a standardized but individualized ramp-bicycle ergometer protocol to reach maximal exercise capacity after 8 - 12 minutes. The exclusively aerobic energy supply during warm-up will be assessed by constant respiratory quotient and constant arterial lactate concentration (<0.5 mmol in the last 5 min). During exercise, respiratory gas exchange, heart rate, blood pressure, ECG and ratings of perceived exertion will be continuously monitored. Venous blood specimens will be collected before exercise (baseline), at the end of the warm-up as well as 2 min, 10 min, and 30 min in recovery. | Diagnostic Test: Performance testing<br>* Performance testing on a bicycle ergometer.<br>|
| Experimental: Recreational athletes, male<br>Bicycle ergometer-based exercise testing. 15 min aerobic warm-up phase (1 W/kg) followed by a standardized but individualized ramp-bicycle ergometer protocol to reach maximal exercise capacity after 8 - 12 minutes. The exclusively aerobic energy supply during warm-up will be assessed by constant respiratory quotient and constant arterial lactate concentration (<0.5 mmol in the last 5 min). During exercise, respiratory gas exchange, heart rate, blood pressure, ECG and ratings of perceived exertion will be continuously monitored. Venous blood specimens will be collected before exercise (baseline), at the end of the warm-up as well as 2 min, 10 min, and 30 min in recovery. | Diagnostic Test: Performance testing<br>* Performance testing on a bicycle ergometer.<br>|
| Experimental: Recreational athletes, female<br>Bicycle ergometer-based exercise testing. 15 min aerobic warm-up phase (1 W/kg) followed by a standardized but individualized ramp-bicycle ergometer protocol to reach maximal exercise capacity after 8 - 12 minutes. The exclusively aerobic energy supply during warm-up will be assessed by constant respiratory quotient and constant arterial lactate concentration (<0.5 mmol in the last 5 min). During exercise, respiratory gas exchange, heart rate, blood pressure, ECG and ratings of perceived exertion will be continuously monitored. Venous blood specimens will be collected before exercise (baseline), at the end of the warm-up as well as 2 min, 10 min, and 30 min in recovery. | Diagnostic Test: Performance testing<br>* Performance testing on a bicycle ergometer.<br>|
| No Intervention: Control, male<br>Venous blood specimens will be collected at the same time points in the absence of exercise. | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Proteomics and autophagy processes | Change in protein levels of autophagy biomarkers (LC3II & p62) of isolated PBMCs (peripheral blood mononuclear cells) by Western Blotting. | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Salivary cortisol levels | Salivary cortisol levels in nmol per Liter (nmol/L) after will be evaluated and compared to the control group | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 
| Proteome patterns | Change in protein levels and protein phosphorylation by untargeted mass spectrometry-based proteomics and phosphoproteomics of isolated PBMCs (peripheral blood mononuclear cells). | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 
| Metabolic processes | Targeted and quantitative analysis by mass spectrometry of change in metabolites of Plasma. | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 
| Lipid profiling | Targeted and quantitative analysis by mass spectrometry of change in plasma Lipids. | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 
| Phosphoproteome patterns | Change in protein phosphorylation by untargeted mass spectrometry-based phosphoproteomics of isolated PBMCs (peripheral blood mononuclear cells). | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 
| Ubiquitinome patterns | Change in protein ubiquitination levels by untargeted mass spectrometry-based proteomics of isolated PBMCs (peripheral blood mononuclear cells). | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 
| Epigenetic patterns | Evaluate epigentic methylation patterns through blood based epigenome analysis | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 
| Exosomal protein patterns | Evaluate exosomal protein content through mass spectrometry based analysis | Before the exercise test (baseline), at the end of the 15 minute warm-up phase and 2 minutes, 10 minutes and 30 minutes after termination of the exercise test. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Exercise Test, Autophagy, Multi-omic, Fitness level