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Skeletal muscle stem cells (Satellite cells) are indispensable for muscle growth and remodeling following myofibril damage. Skeletal muscle trauma is present in numerous catabolic conditions, characterized by elevated proteolysis and muscle wasting such as, cancer cachexia and muscular dystrophy, which result in physical capacity impairment and a deteriorated quality of life. Recent studies performed in animals and cell cultures indicate that the increased levels of inflammation and oxidative stress and the reduction of antioxidant defense may blunt the satellite cells response and myogenic programming during muscle healing. However, evidence regarding the effects of redox status on satellite cells and muscle myogenic potential in humans is lacking. Exercise-induced muscle damage bears striking similarities with the aforementioned conditions, which makes it a valuable tool to investigate the redox-dependent regulation of satellite cells during muscle healing. Thus, the objectives of the present study are to examine the effects of redox status perturbation (via N-acetylcysteine administration) on intracellular pathways responsible for satellite cells responses at rest and following aseptic muscle trauma induced by damaging exercise.
A total number of 40-60 young males aged 18-30 will be initially enrolled in the study. Then, participants will be allocated to either a 1) Low glutathione (GSH-low) or a 2) High glutathione (GSH-high) group based on the basal glutathione levels of their vastus lateralis muscle. In a double-blind, crossover, repeated measures design, participants will consume either Placebo (PLA) or N-acetylcysteine (NAC) before (7-day loading phase), on exercise day and for 8 consecutive days following a single bout of intense exercise (300 eccentric contractions at 30 deg/sec in an isokinetic dynamometer). In both conditions, blood samples and muscle biopsies will be collected at baseline, before the exercise protocol and at 2- and 8-days post-exercise. Muscle performance and soreness will also be assessed at the same time points. Before each trial, participants' dietary intake will be analyzed via diet recalls. Physical activity will be analyzed only at baseline via accelerometry. A 4-week washout period will be implemented between trials. Blood samples will be analyzed for inflammation and oxidative stress markers. Muscle samples will be analyzed for satellite cell responses and myogenic potential, protein levels of intracellular signaling proteins, muscle thiols and antioxidant enzyme activity.
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| Label | Type | Description | Intervention Names |
|---|---|---|---|
| N-Acetylcysteine | Experimental | N-Acetylcysteine supplementation: Orally, 40 mg/kg per day in 3 doses (250 ml each) for 7 consecutive days and immediately post-exercise. The remaining 8 days, 40mg/kg per day in 3 doses (250 ml each). |
|
| Placebo | Active Comparator | Placebo administration: Orally 750 ml per day in 3 doses (250 ml each) for 7 consecutive days and immediately post-exercise. The remaining 8 days, 750 ml per day in 3 doses (250 ml each). |
|
| Name | Type | Description | Arm Group Labels | Other Names |
|---|---|---|---|---|
| N-Acetylcysteine | Dietary Supplement | N-Acetylcysteine in a powder form diluted in a 250 ml drink containing 248 ml water and 2 ml of natural, non-caloric, flavoring-sweetener containing sucralose. |
| Measure | Description | Time Frame |
|---|---|---|
| Change in muscle satellite cells number (i.e. Pax7+ cells) and activation status (i.e. Pax7+/MyoD+ cells) | Satellite cells number and activation status, will be assessed in muscle via immunohistochemistry. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Change in muscle myogenic mRNA expression | mRNA expression levels of Myogenic factor 5 (Myf5), myogenin and Myogenic factor 6 (Myf6/MRF4) and myostatin will be assessed in muscle using Real-Time Polymerase Chain Reaction (RT-PCR). | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Change in muscle inflammatory state | Pro-inflammatory (M1+) and anti-inflammatory (M2+) macrophages will be measured in muscle using immunohistochemistry. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Change in intracellular antioxidant enzymes in muscle | Protein levels of Glutathione peroxidase 3 (GPx3), Superoxide dismutase 1 (SOD1) and Thioredoxin (Trx1) will be measured using western blotting. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Change in muscle thiol content | Concentration levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) will be measured spectophotometrically. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Change in intracellular signaling proteins in muscle | Protein levels of IGF-1, Notch1 and Wnt3 will be measured using western blotting. |
| Measure | Description | Time Frame |
|---|---|---|
| Resting metabolic rate (RMR) | RMR will be assessed after an overnight fast with participants in a supine position following a 15-min stabilization period by taking 30 consecutive 1-min VO2/CO2 measurements using a portable open-circuit indirect calorimeter with a ventilated hood system following a standard calibration protocol. | At baseline |
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Inclusion Criteria:
Exclusion Criteria:
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| Name | Affiliation | Role |
|---|---|---|
| Konstantinos Papanikolaou, PhD | University of Thessaly, School of Physical Education & Sport Science | Principal Investigator |
| Facility | Status | City | State | ZIP | Country | Contacts |
|---|---|---|---|---|---|---|
| Laboratory of Exercise Biochemistry, Exercise Physiology,and Sports Nutrition, School of Physical Education and Sport Science, University of Thessaly | Trikala | Thessaly | 42100 | Greece |
| PubMed Identifier | Type | Citation | Retractions |
|---|---|---|---|
| 36828945 | Derived | Papanikolaou K, Jamurtas AZ, Poulios A, Tsimeas P, Draganidis D, Margaritelis NV, Baloyiannis I, Papadopoulos C, Sovatzidis A, Deli CK, Rosvoglou A, Georgakouli K, Tzatzakis T, Nikolaidis MG, Fatouros IG. Skeletal muscle and erythrocyte redox status is associated with dietary cysteine intake and physical fitness in healthy young physically active men. Eur J Nutr. 2023 Jun;62(4):1767-1782. doi: 10.1007/s00394-023-03102-2. Epub 2023 Feb 24. | |
| 31366396 |
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| ID | Term |
|---|---|
| D000111 | Acetylcysteine |
| ID | Term |
|---|---|
| D003545 | Cysteine |
| D000603 | Amino Acids, Sulfur |
| D013457 | Sulfur Compounds |
| D009930 | Organic Chemicals |
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| Placebo | Dietary Supplement | Placebo consisted of 248 ml water and 2 ml of natural, non-caloric, flavoring-sweetener containing sucralose. |
|
| At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Change in skeletal muscle damage levels | Skeletal muscle damage will be quantified via histochemistry using H&E staining. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Body composition |
Body composition will be measured using a dual-energy x-ray absorptiometry scanner (DXA). |
| At baseline |
| Maximal oxygen consumption (VO2max) | VO2max will be assessed during continuous incremental running to volitional fatigue on a treadmill with a pulmonary gas exchange system (Oxycon Mobile; Sensor-Medics Corporation). | At baseline |
| Isokinetic strength | Maximal knee extensor eccentric peak torque at 60 degrees will be assessed on an isokinetic dynamometer. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Change in delayed onset of muscle soreness (DOMS) | Muscle soreness will assessed during palpation of the muscle belly and the distal region of relaxed vastus medialis, vastus lateralis and rectus femoris following three repetitions of of a full squat. Subjects will rate their DOMS on a visual analogue scale (0-10). | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Physical activity | Level of habitual physical activity will be assessed using accelerometry (ActiGraph GT3X-BT accelerometer). | At baseline. |
| Dietary intake | Daily dietary intake will be assessed using 7-day diet recalls. | At baseline. |
| Interleukin-1β (IL-1β) in blood | Concentration of IL-1β will be measured in plasma. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Interleukin-6 (IL-6) in blood | Concentration of IL-6 will be measured in plasma. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Interleukin-8 (IL-8) in blood | Concentration of IL-6 will be measured in plasma. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Interleukin-10 (IL-10) in blood | Concentration of IL-10 will be measured in plasma. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Cortisol in blood | Concentration of cortisol will be measured in serum. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Protein carbonyls (PC) | Concentration of protein carbonyls will be measured in muscle. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Malondialdehyde (MDA) | Concentration of malondialdehyde will be measured in muscle. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Total antioxidant capacity (TAC) | Concentration of TAC will be measured in serum. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Catalase | Concentration of catalase will be measured in red blood cell lysate. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Creatine Kinase (CK) | Concentration of CK will be measured in serum. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| White blood cell count in blood | White blood cell count will be measured in blood. White blood cell count will be measured in blood. | At baseline, before the exercise protocol and at days 2 and 8 following exercise. |
| Derived |
| Papanikolaou K, Draganidis D, Chatzinikolaou A, Laschou VC, Georgakouli K, Tsimeas P, Batrakoulis A, Deli CK, Jamurtas AZ, Fatouros IG. The redox-dependent regulation of satellite cells following aseptic muscle trauma (SpEED): study protocol for a randomized controlled trial. Trials. 2019 Jul 31;20(1):469. doi: 10.1186/s13063-019-3557-3. |
| D000596 |
| Amino Acids |
| D000602 | Amino Acids, Peptides, and Proteins |